Method for preparation of neo-cartilage constructs

ABSTRACT

Neo-cartilage constructs suitable for implantation into a joint cartilage lesion in situ and a method for repair and restoration of function of injured, traumatized, aged or diseased cartilage. The construct comprises at least chondrocytes incorporated into a support matrix processed according to the algorithm comprising variable hydrostatic or atmospheric pressure or non-pressure conditions, variable rate of perfusion, variable medium composition, variable temperature, variable cell density and variable time to which the chondrocytes are subjected.

This application is a continuation-in-part application of Ser. No.10/104,677 filed on Mar. 22, 2002 and is based on and claims priority ofthe Provisional application Ser. No. 60/425,696 filed on Nov. 12, 2002and Provisional application Ser. No. 60/427,627 filed on Nov. 18, 2002.

BACKGROUND OF THE INVENTION Field of Invention

The current invention concerns neo-cartilage constructs suitable forimplantation into a joint cartilage lesion in situ and a method forrepair and restoration of function of injured, traumatized, aged ordiseased cartilage. The construct of the invention comprises at leastchondrocytes incorporated into a support matrix processed according tothe algorithm of the invention. In particular, the invention concerns aneo-cartilage construct for in situ implanting into a cartilage lesionwherein said construct comprises a cultured differentiated autologous orheterologous chondrocytes or cells which could be differentiated intochondrocytes incorporated into a support matrix and subjected to analgorithm of the invention wherein said algorithm comprises variablehydrostatic or atmospheric pressure or non-pressure conditions, variablerate of perfusion, variable medium composition, variable temperature,variable concentration of oxygen or other gases, variable cell densityand variable time to which the chondrocytes are subjected.

Additionally, the invention concerns a method for generation of theneo-cartilage and the neo-cartilage construct of the invention.

The invention further concerns a method where the implantation of theconstruct of the invention initiates and achieves incorporation of theneo-cartilage into a native surrounding cartilage. The construct isimplanted into the joint cartilage lesion typically below one layer orbetween two layers of biologically acceptable sealants.

The invention further concerns a method for repair and restoration ofthe injured, damaged, diseased or aged cartilage into its fullfunctionality and for treatment of injured or diseased cartilage byimplanting the neo-cartilage construct between two layers ofbiologically acceptable sealants.

BACKGROUND AND RELATED DISCLOSURES

Damage to the articular cartilage which occurs in active individuals andolder generation adults as a result of either acute or repetitivetraumatic injury or aging is quite common. Such damaged cartilage leadsto pain, affects mobility and results in debilitating disability.

Typical treatment choices, depending on lesion and symptom severity, arerest and other conservative treatments, minor arthroscopic surgery toclean up and smooth the surface of the damaged cartilage area, and othersurgical procedures such as microfracture, drilling, and abrasion. Allof these may provide symptomatic relief, but the benefit is usually onlytemporary, especially if the person's pre-injury activity level ismaintained. For example, severe and chronic forms of knee jointcartilage damage can lead to greater deterioration of the jointcartilage and may eventually lead to a total knee joint replacement.Approximately 200,000 total knee replacement operations are performedannually. The artificial joint generally lasts only 10 to 15 years andthe operation is, therefore, typically not recommended for people underthe age of fifty.

It would, therefore, be extremely advantageous to have available amethod for in situ treatment of these injuries which would effectivelyrestore the cartilage to its pre-injury state.

Attempts to provide means and methods for repair of articular cartilageare disclosed, for example, in U.S. Pat. Nos. 5,723,331; 5,786,217;6,150,163; 6,294,202; 6,322,563 and in the U.S. patent application Ser.No. 09/896,912, filed on Jun. 29, 2001.

U.S. Pat. No. 5,723,331 describes methods and compositions forpreparation of synthetic cartilage for the repair of articular cartilageusing ex vivo proliferated denuded chondrogenic cells seeded ex vivo, inthe wells containing adhesive surface. These cells redifferentiate andbegin to secrete cartilage-specific extracellular matrix therebyproviding an unlimited amount of synthetic cartilage for surgicaldelivery to a site of the articular defect.

U.S. Pat. No. 5,786,217 describes methods for preparing a multi-celllayered synthetic cartilage patch prepared essentially by the samemethod as described in '331 patent except that the denuded cells arenon-differentiated, and culturing these cells for a time necessary forthese cells to differentiate and form a multi cell-layered syntheticcartilage.

U.S. application Ser. No. 09/896,912, filed on Jun. 29, 2001 concerns amethod for repairing cartilage, meniscus, ligament, tendon, bone, skin,cornea, periodontal tissues, abscesses, resected tumors and ulcers byintroducing into tissue a temperature dependent polymer gel inconjunction with at least one blood component which adheres to thetissue and promotes support for cell proliferation for repairing thetissue.

None of the above cited references results in repair and regeneration ofcartilage in situ including de novo formation of the superficialcartilage layer sealing a joint cartilage lesion in situ.

It is thus a primary objective of this invention to provide a method andmeans for regeneration of injured or traumatized cartilage by forming,in the injured lesion of the cartilage, a cavity, by administering atleast one but typically two separate layers of a biologically acceptableglue sealant and implanting a neo-cartilage containing construct underthe one layer or into said cavity. The method according to the inventionresults in the growth of the superficial cartilage layer over the lesionand sealing the lesion.

All patents, patent applications and publications cited herein arehereby incorporated by reference.

SUMMARY

One aspect of the current invention is a neo-cartilage constructsuitable for implantation into a cartilage lesion in situ.

Another aspect of the current invention is a neo-cartilage constructimplanted under one or between two layers of biologically acceptablesealants within a cartilage lesion.

Another aspect of the current invention is a method for fabrication of athree-dimensional neo-cartilage construct of the invention comprisingsteps of:

a) preparing a support matrix structure;

b) harvesting a piece of cartilage from a donor for isolation ofchondrocytes;

c) culturing and expanding the chondrocytes;

d) suspending the expanded chondrocytes in a suspension fluid;

e) incorporating said suspended chondrocytes into said matrix; and

f) propagating said chondrocytes into two or three-dimensionalneo-cartilage construct using the algorithm of the invention.

Still another aspect of the current invention is a method for generationof an autologous type of neo-cartilage construct by generating a carriersupport for autologous chondrocytes cultured into neo-cartilage whereinsaid support is a biologically acceptable cell-carrier thermo-reversiblepolymer gel or a thermo-reversible gelation hydrogel (CCTG, TRGH orVITROGEN®), wherein said neo-cartilage is suspended within the CCTG andwherein a resulting CCTG/neo-cartilage or TRGH/neo-cartilage or asuspension thereof is injected into the cartilage lesion.

Still another aspect of the current invention is a neo-cartilageconstruct implanted in situ into a cartilage lesion between two layersof sealants wherein a first sealant is deposited at the bottom of acartilage lesion and the second sealant is deposited over the implantedconstruct on the top of the cartilage lesion and wherein the secondsealant leads to formation and growth of superficial cartilage layerwhich seals said cartilage lesion.

Another aspect of the current invention is a method for repair andrestoration of damaged, injured, diseased or aged cartilage to afunctional cartilage, said method comprising steps:

a) preparing a neo-cartilage construct comprising autologous orheterologous chondrocytes incorporated into a sponge, porous scaffold orthermo-reversible gelation hydrogel (TRGH) matrix support and subjectedto the algorithm of the invention;

b) optionally introducing a first layer of a first biologicallyacceptable sealant into a cartilage lesion;

c) implanting said construct into said lesion or into said cavity overthe first layer of said first sealant;

d) introducing a second layer of a second biologically acceptablesealant over said construct wherein said second sealant may or may notbe the same as the first sealant and wherein a combination of saidconstruct and said second sealant results in formation and growth of asuperficial cartilage layer sealing the cartilage lesion in situ.

Another aspect of the current invention is a method for repair andrestoration of damaged, injured, diseased or aged cartilage to afunctional cartilage, said method comprising steps:

a) obtaining autologous or heterologous chondrocytes;

b) culturing said chondrocytes ex vivo into a neo-cartilage, saidneo-cartilage comprising autologous or heterologous chondrocytesincorporated into a sponge or TRGH matrix support subjected to thealgorithm of the invention;

c) optionally introducing a first layer of a first biologicallyacceptable sealant into a cartilage lesion;

d) depositing a space-holding thermo-reversible gel (SHTG) or TRGH intoa lesion or into a cavity formed above the first sealant layer therebypermitting sufficient time for growth and differentiation of ex vivocultured neo-cartilage, said space holding thermo-reversible gel (SHTG)deposited into said cavity as a sol at temperatures between about 5 toabout 25° C., wherein within said cavity and at the body temperaturesaid SHTG converts from the fluidic sol into a solid gel and in thisform SHTG holds the space for subsequent introduction of theneo-cartilage cultured ex vivo, and provides protection against cell andblood-borne agents migration into the cavity from the subchondral spaceand from the synovial capsule and wherein its presence further providesa substrate for and promotes in situ formation of a de novo superficialcartilage layer covering the cartilage lesion;

e) depositing a second layer of a second biologically acceptable sealantover the cartilage lesion;

f) removing said SHTG by cooling said lesion to change SHTG into sol;

f) depositing said neo-cartilage cultured ex vivo into the cavity formedbetween two layers of sealants and under the de novo formed superficialcartilage layer;

g) removing said SHTG or TRGH from the cavity after the neo-cartilageintegration into a native cartilage under the formed superficialcartilage layer by cooling said lesion to from about 5 to about 15° C.to convert the solid gel into fluidic sol and removing said sol or, inalternative, leaving said SHTG or TRGH to disintegrate and be removednaturally.

Another aspect of the current invention is a method for repair andrestoration of damaged, injured, diseased or aged cartilage to afunctional cartilage, said method comprising steps:

a) preparing an intact and discreet piece of neo-cartilage by culturingautologous or heterologous chondrocytes ex vivo, suspending saidcultured chondrocytes in a thermo-reversible gelation hydrogel (TRGH)and warming said suspension of chondrocytes to temperature above 30° C.in order to convert TRGH into a solid gel and subjecting the solid gelto the algorithm of the invention;

b) introducing a first and a second layer of a first and a secondbiologically acceptable sealant into a cartilage lesion;

c) cooling said TRGH/neo-cartilage to 5-15° C. to sol state;

d) depositing said neo-cartilage suspended in the TRGH into a cavityformed between two layers of sealants as a sol at temperatures betweenabout 5 to about 25° C. wherein, within said cavity and at the bodytemperature, said TRGH converts from the sol state into the solid geland in this state provides protection for and enables integration ofdeposited neo-cartilage into a native surrounding cartilage and whereinthe presence of TRGH further provides a substrate and promotes in situformation of de novo superficial cartilage layer covering the cartilagelesion; and

e) leaving said TRGH in the lesion until its disintegration or, inalternative, removing said TRGH from the cavity as a sol by cooling saidlesion to temperature between 5 and 15° C. after the neo-cartilageintegration and formation of superficial cartilage layer.

Another aspect of the current invention is a method for repair andrestoration of damaged, injured, diseased or aged cartilage to afunctional cartilage, said method comprising steps:

a) preparing neo-cartilage or a neo-cartilage containing constructcomprising autologous cultured chondrocytes incorporated into a gel orthermo-reversible gel matrix support ex vivo and subjected to thealgorithm of the invention;

b) introducing a first layer of a first biologically acceptable sealantinto a cartilage lesion;

c) depositing said construct or said neo-cartilage over the first layerof the first sealant; and

d) depositing a layer of a second biologically acceptable sealant eitherover the neo-cartilage construct or the neo-cartilage deposited into acartilage lesion and covering the lesion with said second sealant,wherein in time said neo-cartilage is integrated into the nativecartilage and wherein the presence of the neo-cartilage construct andthe second sealant promotes in situ formation and growth of de novosuperficial cartilage layer covering the cartilage lesion.

Still another aspect of the current invention is a method for generationand maintaining integrity of the lesion cavity for the introduction ofneo-cartilage, a neo-cartilage gel, a neo-cartilage suspension orneo-cartilage construct from a synovial capsule and for blocking themigration of subchondral and synovial cells and cell and blood productsinto said cavity and for providing a substrate for a formation ofsuperficial cartilage layer overgrowing the lesion by introducing abiologically acceptable space-holding thermo-reversible gel (SHTG) intoa cleaned lesion for a duration of culturing autologous chondrocytesinto neo-cartilage before introducing said neo-cartilage orneo-cartilage construct or suspension into the lesion.

Still another aspect of the current invention is a method for treatmentof damaged, injured, diseased or aged cartilage by utilizing any of themethods listed above to implant the neo-cartilage construct into thelesion.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a construct comprising neo-cartilage. FIG. 1A is aschematic drawing of the sponge made of sol/gel showing the distributionof chondrocytes within the collagen sponge. FIG. 1B is a micrograph ofthe actual neo-cartilage construct held in the forceps having 4 mm indiameter and thickness of 1.5 mm. Seeding density of the construct is300,000 chondrocytes per 25 μl of collagen solution (12,000,000cells/ml).

FIG. 2A shows a diagram of hydrostatic pressure culture system. FIG. 2Bshows a TESS culture processor unit.

FIG. 3A is a graph representing S-GAG accumulation in cell constructssubjected to static atmospheric (control) or cyclic hydrostatic pressure(test). FIG. 3B is a photomicrograph of Safranin-O staining for S-GAG onparaffin sections in 18 days subjected to static pressure. FIG. 3C is aphotomicrograph of Safranin-O staining for S-GAG on paraffin sections incell constructs subjected to cyclic hydrostatic pressure for 6 daysfollowed by 12 days of static pressure.

FIG. 4 illustrates effect of cyclic and constant hydrostatic pressure onproduction of S-GAG (FIG. 4A) and DNA (FIG. 4B).

FIG. 5A shows S-GAG accumulation in cell constructs under continuedculture conditions of static culture (control), medium perfusion(COMPa), cyclic hydrostatic pressure (Cy-HP) combined with mediumperfusion (control) and constant hydrostatic pressure combined withmedium perfusion (constant-HP). FIG. 5B illustrates DNA content at day 6and day 18 in cells constructs submitted to static conditions (control),medium perfusion only (COMPa), cyclic hydrostatic pressure (Cy-HP) andconstant hydrostatic pressure (constant-HP).

FIG. 6A is a photomicrograph of Safranin-O staining for S-GAG onparaffin sections in 18 days cell constructs subjected to staticatmospheric pressure. FIG. 6B is a photomicrograph of Safranin-Ostaining for S-GAG on paraffin sections in cell constructs subjected tocyclic hydrostatic pressure for 6 days followed by 12 days of staticpressure. FIG. 6C is a photomicrograph of type II collagenimmunohistochemistry on paraffin sections in 6 days cell constructssubjected to static atmospheric pressure. FIG. 6D is a photomicrographof type II collagen immunohistochemistry on paraffin sections in cellconstructs subjected to cyclic hydrostatic pressure for 6 days.

FIG. 7A is a graph illustrating effect of the medium perfusion flow rateon cell proliferation (DNA content) by cell constructs subjected to amedium flow rate of either 0.005 or 0.05 ml/min. FIG. 7B illustrateseffect of flow rate on production of S-GAG.

FIG. 8 shows accumulation detected histologically by toluidine S-GAGblue staining after 15 days culture submitted to perfusion (FIG. 8A),cyclic hydrostatic pressure (FIG. 8B) and constant hydrostatic pressure(FIG. 8C).

FIG. 9 illustrates effect of low oxygen tension on S-GAG production(FIG. 9A) and cell proliferation (FIG. 9B).

FIG. 10A shows an arthroscopic observation of the control empty defectsite 2 weeks after creating empty defect. FIG. 10B shows an arthroscopicobservation of the porcine neo-cartilage (Porcine-NeoCart™) implant site2 weeks after the implantation.

FIG. 11 shows the control lesion without treatment with porcineneo-cartilage where the proliferation of fibrocartilage within thedefect site is clearly visible after 4 months. FIG. 11A shows a defectsite vis-a-vis subchondral bone with a site of formation offibrocartilage. FIG. 11B shows a defect site synovium and synovialmigration. FIG. 11C shows the defect site and formation offibrocartilage.

FIGS. 12A and 12B shows integration of porcine neo-cartilage into thelesion within the host's cartilage after 3 months. FIG. 12C shows theregenerated hyaline-like cartilage in the porcine neo-cartilageimplanted site. FIG. 12D shows the integration between the porcineneo-cartilage and the host cartilage laterally and at the subchondralbone.

FIG. 13A shows S-GAG production in cell constructs subjected to cyclichydrostatic pressure and to atomospheric pressure (control) with mediumperfusion. FIG. 13B shows DNA content in cell constructs subjected tocyclic and constant hydrostatic pressure with medium perfusion.

FIG. 14 shows histological evaluation of cell constructs by Safranin-O.FIG. 14A shows S-GAG accumulation at day 0 (initial). FIG. 14B showsaccumulation of S-GAG on day 21 in cell constructs subjected toatmospheric pressure (control). FIG. 14C shows accumulation of S-GAG onday 21 in cell constructs subjected to 7 days of cyclic hydrostaticpressure (Cy-HP#1) followed by 14 days of to atmospheric pressure. FIG.14D shows accumulation of S-GAG on day 21 in cell constructs subjectedto 14 days of cyclic hydrostatic pressure (Cy-HP#2) followed by 7 daysof to atmospheric pressure. FIG. 14E shows accumulation of S-GAG on day21 in cell constructs subjected to 7 days of constant hydrostaticpressure (Constant-HP) followed by 14 days of atmospheric pressure.

DEFINITIONS

As used herein:

“Chondrocyte” means a nondividing cartilage cell which occupies a lacunawithin the cartilage matrix.

“Isogenous chondrocytes” means clones of cartilage cell derived from onecell of division. Isogenous chondrocytes occur in clusters calledisogenous nests.

“Autologous chondrocytes” means chondrocytes isolated from a donor's ownhealthy articular cartilage.

“Heterologous chondrocytes” means chondrocytes derived from a donor of adifferent species or from a donor of the same species but not therecipient individual or a donor tissue that is derived from therecipient individual but is non-articular cartilage isolated from acartilage of the different species.

“Support matrix” means biologically acceptable sol-gel or spongescaffold suitable for seeding expanded chondrocytes that provides astructural support for growth and three-dimensional propagation ofchondrocytes. The support matrix is prepared from such materials as TypeI collagen, Type II collagen, Type IV collagen, gelatin, agarose,cell-contracted collagen containing proteoglycans, glycosaminoglycans orglycoproteins, fibronectin, laminin, bioactive peptide growth factors,cytokines, elastin, fibrin, synthetic polymeric fibers made ofpoly-acids such as polylactic, polyglycolic or polyamino acids,polycaprolactones, polyamino acids, polypeptide gel, copolymers thereofand combinations thereof. The gel solution matrix may be a polymericthermo-reversible gelling hydrogel. The support matrix is preferablybiocompatible, biodegradable, hydrophilic, non-reactive, has a neutralcharge and be able to have or has a defined structure.

“Neo-cartilage” means an immature hyaline cartilage wherein the ratio ofextracellular matrix to chondrocytes is lower than in mature hyalinecartilage.

“Mature hyaline cartilage” means cartilage consisting of groups ofisogenous chondrocytes located within lacunae cavities which arescattered throughout an extracellular collagen matrix.

“Autologous Cultured Neo-Cartilage” means a hyaline neo-cartilage tissuegrown ex vivo from chondrocytes isolated from a donor's own healthyarticular cartilage.

“Neo-cartilage construct”, “NEOCART™” or “NeoCart™” means a3-dimensional structural composition comprising chondrocytesincorporated into a matrix support treated by or subjected to thealgorithm of the invention. Neo-cartilage construct thus means adiscrete piece of hyaline neo-cartilage formed from culturedchondrocytes for implantation into lesion of a damaged, aged or diseasedcartilage wherein, after implantation, the neo-cartilage is integratedinto a native cartilage within the lesion. NeoCart® cartilage ismanufactured by and is proprietary of Histogenics Corporation,Easthampton, Mass.

“TESS™” means Tissue Engineering Support System which is available asTESS culture processor unit for culturing of chondrocytes prepared fromarthroscopic biopsy samples. The unit permits changes in hydrostaticpressure, including cyclic hydrostatic pressure changes and controlsother physical parameters such as temperature, gas concentration, mediumperfusion rate and such other parameters as may be needed. Relevantdetailed information is found in U.S. Pat. No. 6,432,713 B2, patentapplication Ser. No. 09/895,162, Ser. No. 09/895,161, PCT JP01/01516,Japanese patent applications 2001-126543 and 2001-261556, incorporatedherein by reference.

“Sealant” means a biologically acceptable typically rapid-gellingformulation having a specified range of adhesive and cohesiveproperties. Sealant is thus a biologically acceptable rapidly gellingsynthetic compound having adhesive and/or gluing properties, and istypically a hydrogel, such as derivatized polyethylene glycol (PEG)which is preferably cross-linked with a collagen compound, typicallyalkylated collagen. Examples of suitable sealants aretetra-hydrosuccinimidyl or tetra-thiol derivatized PEG, or a combinationthereof, commercially available from Cohesion Technologies, Palo Alto,Calif. under the trade name CoSeal™, described in J. Biomed. Mater. ResAppl. Biomater., 58:545-555 (2001), or two-part polymer compositionsthat rapidly form a matrix where at least one of the compounds ispolymeric, such as, polyamino acid, polysaccharide, polyalkylene oxideor polyethylene glycol and two parts are linked through a covalent bond,as described in U.S. Pat. No. 6,312,725B1, herein incorporated byreference, and cross-linked PEG with methyl collagen, such as across-linked polyethylene glycol hydrogel with methyl-collagen. Thesealant of the invention typically gels and/or bonds rapidly uponcontact with tissue, particularly with tissue containing collagen.

“First sealant” means a biologically acceptable tissue sealant which isdeposited at the bottom of the lesion.

“Second sealant” means a biologically acceptable sealant which isdeposited above and over the neo-cartilage construct implanted into alesion. The second sealant may or may not be the same as the firstsealant and is preferably a cross-linked polyethylene glycol hydrogelwith methyl-collagen.

“Hydrostatic pressure” means pressure measured above the atmosphericpressure.

“Cyclic hydrostatic pressure” or “Cy-HP” means the application ofrepeated, two or multiplicity periods of applied hydrostatic pressurewithin a defined loading interval which creates a sine wave form ofmeasured pressure.

“Constant hydrostatic pressure”, “constant-HP” or “CHP” means theapplication of a non-fluctuating or non-cyclic pressure load over aperiod of time.

“Loading” or “loading interval” means a period of applied cyclichydrostatic pressure load followed by a return to atmospheric pressurewhere no external pressure is applied.

“Resting phase” means a variable length of time wherein cells aremaintained in culture at atmospheric pressure after exposure to orculturing under cyclic hydrostatic pressure.

“De novo” or “de novo formation” means the new production of cells, suchas chondrocytes, fibroblasts, fibrochondrocytes, tenocytes, osteoblastsand stem cells capable of differentiation, or tissues such as cartilageconnective tissue, fibrocartilage, tendon, and bone within a supportstructure, such as multi-layered system, scaffold or collagen matrix orformation of superficial cartilage layer.

“Superficial cartilage layer” means an outermost layer of cartilage thatforms the layer of squamous-like flattened superficial zone chondrocytescovering the layer of the second sealant and overgrowing the lesion.

“Thermo-reversible” means a compound or composition changing itsphysical properties such as viscosity and consistency, from sol to gel,depending on the temperature. The thermo-reversible composition istypically completely in a sol (liquid) state at between about 5 and 15°C. and in a gel (solid) state at about 30° C. and above. The gel/solstate in between shows a lesser or higher degree of viscosity anddepends on the temperature. When the temperature is higher than 15° C.,the sol begins to change into gel and with the temperature closer to30-37° the sol becomes more and more solidified as gel. At lowertemperatures, typically lower than 15° C., the sol has more liquidconsistency.

“TRGH” means thermo-reversible gelation hydrogel material in which thesol-gel transition occurs on the opposite temperature cycle of agar andgelatin gels. Consequently, the viscous fluidic phase is in a sol stageand the solid phase is in a gel stage. TRGH has very quick sol-geltransformation which requires no cure time and occurs simply as afunction of temperature without hysteresis. The sol-gel transitiontemperature can be set at any temperature in the range from 5° C. to 70°C. by molecular design of thermo-reversible gelation polymer (TGP), ahigh molecular weight polymer of which less than 5 wt % is enough forhydrogel formation.

“SHTG” means space holding thermo-reversible gel.

“Sol-gel solution” means a colloidal suspension which, under certainconditions, transitions from a liquid (sol) to a solid material (gel).The “sol” is a suspension of aqueous collagen that is transitioned, byheat treatment, into a gel.

“GAG” means glycosaminoglycan.

“S-GAG” means sulfated glycosaminoglycan.

“MMP” means matrix metalloproteinase, an enzyme associated withcartilage degeneration in an injured or diseased joint.

“DMB” means dimethylene blue used for staining of chondrocytes.

“MPa” means MegaPascal. One MPa is equal to 145 psi.

“Superficial zone cartilage” means the flattened outermost layer ofchondrocytes covering the extracellular matrix intermediate zone anddeeper zone of mature articular cartilage in which non-dividing cellsare dispersed.

“Connective tissue” means tissue that protect and support the bodyorgans, and also tissues that hold organs together. Examples of suchtissues include mesenchyme, mucous, connective, reticular, elastic,collagenous, bone, blood, or cartilage tissue such as hyaline cartilage,fibrocartilage, and elastic cartilage.

“The algorithm” means variable defined conditions, such as variablepressure or non-pressure conditions, variable perfusion rate, differentmedium, different cell density, different temperature, variable time,different oxygen and carbon dioxide conditions, etc., to which acellular construct of neo-cartilage is subjected in order to convert itto a mature neo-cartilage construct.

“Adhesive strength” means a peel bond strength measurement, which can beaccomplished by bonding two plastic tabs with an adhesive formulation.The tabs can be formed by cutting 1×5 cm strips from polystyreneweighing boats. To the surface of the boat are bonded (using commercialcyanoacrylate Superglue), sheets of sausage casing (collagen sheeting,available from butcher supply houses). The sausage casing is hydrated inwater or physiological saline for 20 min to one hour and the adhesive isapplied to a 1×1 cm area at one end of the tab; the adhesive is cured.Then, the free ends of the tab are each bent and attached to the upperand lower grips, respectively, of a tensile testing apparatus and pulledat 10 mm/min strain rate, recording the force in Newtons to peel. Aconstant force trace allows estimation of N/m, or force per width of thestrip. A minimum force per width of 10 N/m is desired; 100 N/m or higheris more desirable. Alternatively, the same tab can be bonded (a singletab) over a 1×1 cm area to tissue, either dissected or exposed tissue ina living animal, during surgery. The free end of the tab is then grippedor attached through a perforation to a hook affixed to a hand-heldtensile test device (Omega DFG51-2 digital force gauge; OmegaEngineering, Stamford, Conn.) and pulled upward at approximately 1cm/sec. The maximum force required to detach the tab from the tissue isrecorded. The minimum force desired in such measurements would be 0.1 Nto detach the tab. Forces or 0.2 to 1 N are more desirable.

“Cohesive strength” means the force required to achieve tensile failureand is (pulling in extension); measured using a tensile test apparatus.The glue or adhesive can be cured in a “dog-bone”-shaped mold. The wideends of the formed solid adhesive can then be affixed, usingcyanoacrylate (Superglue) to plastic tabs, and gripped in the testapparatus. Force at extensional failure should be at least 0.2 MPa (2N/cm2) but preferably 0.8 to 1 MPa or higher.

“Lap shear measurements” means a test of bonding strength, in which thesealant formulation is applied to overlapping tabs of tissue, cured, andthen the force to pull the tabs apart is measured. The test reflectsadhesive and cohesive bonding; strong adhesives will exhibit values of0.5 up to 4-6 N/cm² of overlap area.

DETAILED DESCRIPTION OF THE INVENTION

This invention is based on finding that when metabolically active butnon-dividing chondrocytes are processed according to the invention, theybecome activated and dividing. These activated and dividing chondrocytesthen may be converted into neo-cartilage and upon incorporating thisneo-cartilage into the support matrix and submitting saidneo-cartilage/support matrix to the algorithm of the invention become astructural unit called neo-cartilage construct. Such processedneo-cartilage construct is suitable for implantation into a lesion ofinjured, traumatized, aged or diseased cartilage or under the topsealant or between layers of a first (bottom) and a second (top)sealant. Under these conditions the second top sealant promotes in situformation of de novo superficial cartilage layer over the cartilagelesion.

The invention thus, in its broadest scope, concerns a method forpreparation of neo-cartilage from chondrocytes harvested from a donor'stissue, a method for formation of a support matrix, a method forfabrication of a neo-cartilage construct, a method for de novo formationof a superficial cartilage layer in situ, a method for repair andrestoration of damaged, injured, traumatized or aged cartilage to itsfull functionality, and a method for treatment of injuries or diseasescaused by damaged cartilage due to the trauma, injury, disease or age.

Briefly, the invention comprises preparation of neo-cartilage fromharvested autologous or heterologous chondrocytes, culturing andexpansion of chondrocytes, seeding the chondrocytes within a collagenousor thermo-reversible gel support matrix and propagating saidchondrocytes in two or three-dimensions. To achieve the chondrocytepropagation, the seeded support matrix is optionally subjected to thealgorithm of variable conditions, such as static conditions, constant orcyclic hydrostatic pressure, temperature changes, oxygen and/or carbondioxide level changes and changes in perfusion flow rate of the culturemedium in the presence of various supplements, such as, growth factors,donor's serum, ascorbic acid, ITS, etc. The chondrocyte-seeded supportmatrix treated as above becomes a neo-cartilage construct(neo-cartilage) suitable for implanting into a joint cartilage lesion.

The neo-cartilage construct is implanted into the lesion under a topsealant, or into a cavity formed by two layers of adhesive sealants. Thefirst layer of the sealant is deposited at and covers the bottom of thelesion and its function is to protect the integrity of said lesion fromcell migration and from effects of various blood and tissue metabolitesand also to form a bottom of the cavity into which the neo-cartilageconstruct is deposited.

In one embodiment, after the neo-cartilage construct is emplaced intothe lesion cavity, the second adhesive layer is deposited on the top ofthe neo-cartilage construct and within several months results information of the superficial cartilage layer completely sealing thelesion.

In the alternative embodiment, two adhesive layers may be depositedconcurrently with or before the construct is implanted into the cavitybetween them. In such an instance, in the interim, said cavity may befiled with a space holding thermo-reversible gel (SHTG). Both sealantlayers and the construct or space holding gel are left within the lesioncavity for a certain predetermined period of time, typically from oneweek to several months, or in case of the space holding gel, until theneo-cartilage construct is prepared ex vivo and ready to be implanted.The second layer deposited on the top and over the lesion promotesformation of a superficial cartilage layer which covers the lesion onthe outside and eventually overgrows the lesion completely therebyresulting in complete or almost complete sealing of the lesion and ofthe neo-cartilage construct deposited within said lesion leading toincorporation of neo-cartilage into a native cartilage and resulting inhealing of the injured or damaged cartilage. In alternative, thethermo-reversible gel may serve as an initiator for promotion offormation of the superficial cartilage layer.

Both the support matrix of the neo-cartilage construct or the spaceholding thermo-reversible gel deposited into the lesion are materialswhich are biodegradable and permit and promote formation of thesuperficial cartilage layer and integration of the chondrocytes from theneo-cartilage construct into the native cartilage within the lesioncavity. Such integration begins within several weeks or months followingthe implanting and may continue for several months and involves a growthand maturing of neo-cartilage into normal cartilage integrated into thehealthy cartilage. The top sealant layer promotes an overgrowth of thelesion with the superficial cartilage layer typically in about two-threemonths when the sealant is itself degraded.

In the alternative embodiment, the lesion cavity is filled with aspace-holding gel until the outer superficial cartilage layer is formedat which time the neo-cartilage construct comprising ex vivo propagatedchondrocytes suspended in a thermo-reversible sol is introduced at atemperature between 5° and 15° C. After it is introduced into the lesionas a liquid sol, the introduced thermo-reversible sol-gel is convertedinto a solid gel at body temperatures of 37° C. or at the same orsimilar temperature as the temperature of the synovial cavity. Theneo-cartilage construct introduced into the lesion is integrated intothe native cartilage surrounding the cavity and is completely coveredwith the superficial cartilage layer.

In the alternative, the neo-cartilage construct is deposited into alesion of injured, traumatized, aged or diseased cartilage over thefirst (bottom) sealant layer and the thermo-reversible gel of theneo-cartilage construct promotes in situ formation of the superficialmembrane without a need to add the second sealant.

The method for treatment of injured, traumatized, diseased or agedcartilage comprises treating the injured, traumatized, diseased or agedcartilage with an implanted neo-cartilage construct prepared by methodsdescribed above and/or by any combination of steps or components asdescribed.

I. Preparation of Neo-Cartilage Constructs

Preparation of neo-cartilage constructs for implanting into thecartilage lesion involves harvesting and culturing chondrocytes, seedingthem in the support matrix and preparation thereof, and propagating thechondrocytes either ex vivo, in vitro, or in vivo.

A. Cartilage and Neo-Cartilage

Cartilage is a connective tissue covering joints and bones.Neo-cartilage is immature cartilage which eventually, upon depositioninto the lesion according to this invention, is integrated into andacquires properties of mature cartilage. Differences between the twotypes of cartilage is in their maturity. Cartilage is a mature tissuecomprising metabolically active but non-dividing chondrocytes;neo-cartilage is an immature cartilage comprising metabolically andgenetically activated chondrocytes which are able to divide andmultiply. This invention utilizes properties of neo-cartilage inachieving repair and restoration of damaged cartilage into the fullfunctionality of the healthy cartilage by enabling the neo-cartilage tobe integrated into the mature cartilage surrounding the lesion and inthis way repair the defect.

a) Cartilage

Cartilage is a connective tissue characterized by its poor vascularityand a firm consistency. Cartilage consist of mature non-dividingchondrocytes (cells), collagen (interstitial matrix of fibers) and aground proteoglycan substance (glycoaminoglycans ormucopolysaccharides). Later two are cumulatively known as extracellularmatrix.

There are three kinds of cartilage, namely hyaline cartilage, elasticcartilage and fibrocartilage. Hyaline cartilage found primarily injoints has a frosted glass appearance with interstitial substancecontaining fine type II collagen fibers obscured by proteoglycan.Elastic cartilage is a cartilage in which, in addition to the collagenfibers and proteoglycan, the cells are surrounded by a capsular matrixsurrounded by an interstitial matrix containing elastic fiber network.The elastic cartilage is found, for example, in the central portion ofthe epiglottis. Fibrocartilage contains Type I collagen fibers and istypically found in transitional tissues between tendons, ligaments orbones.

The articular cartilage of the joints, such as the knee cartilage, isthe hyaline cartilage which consists of approximately 5% of chondrocytes(total volume) seeded in approximately 95% extracellular matrix (totalvolume). The extracellular matrix contains a variety of macromolecules,including collagen and proteoglycan. The structure of the hyalinecartilage matrix allows it to reasonably well absorb shock and withstandshearing and compression forces. Normal hyaline cartilage has also anextremely low coefficient of friction at the articular surface.

Healthy hyaline cartilage has a contiguous consistency without anylesions, tears, cracks, ruptures, holes or shredded surface. Due totrauma, injury, disease such as osteoarthritis, or aging, however, thecontiguous surface of the cartilage is disturbed and the cartilagesurface shows cracks, tears, ruptures, holes or shredded surfaceresulting in cartilage lesions. Partly because hyaline cartilage isavascular, the spontaneous healing of large defects is not believed tooccur in humans and other mammals and the articular cartilage has thusonly a limited, if any, capacity for repair.

A variety of surgical procedures have been developed and used inattempts to repair damaged cartilage. These procedures are performedwith the intent of allowing bone marrow cells to infiltrate the defectand promote its healing. Generally, these procedures are only partlysuccessful. More often than not, these procedures result in formation ofa fibrous cartilage tissue (fibrocartilage) which does fill and repairthe cartilage lesion but, because it is qualitatively different beingmade of Type I collagen fibers, it is less durable and less resilientthan the normal articular (hyaline) cartilage and thus has only alimited ability to withstand shock and shearing forces than does healthyhyaline cartilage. Since all diarthroid joints, particularly kneesjoints, are constantly subjected to relatively large loads and shearingforces, replacement of the healthy hyaline cartilage with fibrocartilagedoes not result in complete tissue repair and functional recovery.

b) Neo-Cartilage

Neo-cartilage is an immature hyaline cartilage where the ratio ofextracellular matrix to chondrocytes is lower than in mature hyalinecartilage. Mature hyaline cartilage has the ratio of the extracelluarmatrix to chondrocytes approximately 95:5. The neo-cartilage has a lowerratio of the extracelluar matrix to chondrocytes than mature cartilageand thus comprises more than 5% of chondrocytes.

In the process of development of this invention, it was discovered thatunder the conditions described below, the older inactive chondrocytescould be activated from static non-dividing stage to an active stagewhere they divide, multiply, promote growth of the extracellular matrixand develop into new cartilage (neo-cartilage). The neo-cartilage thuscontains chondrocytes which were rejuvenated and are surrounded by anewly synthesized extracellular-matrix macromolecules. A process foractivation was found to require certain period of time, typically fromabout 1 week to about 3 months and it is thus preferred that theneo-cartilage be prepared ex vivo where nutrients needs and mechanicalloading are well defined.

B. Preparation of Neo-Cartilage

Neo-cartilage prepared according to the current invention is grown exvivo from chondrocytes isolated from the mammalian donor's source. Inthe alternative, neo-cartilage may also be grown in situ or in vivounder conditions described below.

Typical donor sources of mammalian chondrocytes are swine or humans.Neo-cartilage of the invention for human use is preferably grown fromautologous chondrocytes obtained from the patient during arthroscopy.While it is preferred that for human use chondrocytes are autologous, itis to be understood that chondrocytes obtained from other mammaliansources are equally suitable for preparation of neo-cartilage fortreatment of damaged, diseased or aged cartilage. The use of bothautologous and heterologous chondrocytes is intended to be within thescope of the invention.

a) Isolation of Chondrocytes

Specific procedures used for isolation of mammalian chondrocytesgenerally using swine cartilage as an example are described inExample 1. The isolation of human chondrocytes and preparation ofautologous human neo-cartilage is according to procedures described inExample 2.

Briefly, the donor cartilage is obtained either by arthroscopic biopsyfrom the human donor or from a joint or bone, such as, for example, thefemur of the slaughtered animal and processed according to Example 1 or2. The cartilage is preferably digested by collagenase, a strongprotease, most preferably Type I collagenase, in a solution containingpreferably about 0.15% of collagenase. The digestion is run for severalhours to several days, preferably for about 18 hours.

In alternative, the extracellular matter can be digested with proteasesor sugar lyases including but not limited to heparitinase, heparinase,chondroitinase ABC, chondroitinase B and chondroitinase AC. The lyasesare added in admixture with collagenase or in a sequential enzymedigestion steps. These lyases promote further isolation of thechondrocytes from the extracellular matrix (ECM) including disruptionthe glycosaminoglycans of the pericellular environment such that thechondrocytes do not receive inhibitory signals that prevent them fromdividing or producing healthy new extracellular matrix. This finding isespecially important for osteoarthritic chondrocytes which have veryslow division rates and reduced ability to produce extracellular matrix.

This is especially important for osteoarthritic chondrocytes which havevery slow division rates and reduced ability to produce ECM. U.S. Pat.No. 5,916,557 shows that application of chondroitinase ABC tochondrocytes in vitro resulted couterintuitively in the promotion of newcartilage production.

The ability to free the chondrocytes from all extra- and pericellularinhibitory material and thereby to promote cell expansion anddifferentiation is especially important in autologous osteoarthritictissue where the growth is otherwise slow because these chondrocyteshave reduced ability to produce ECM where neo-cartilage formation in theTESS processor under pressure is greatly improved by this early step ofthe process. Furthermore, this method of stimulating chondrocyte growthand differentiation is relatively benign compared to the application ofgrowth factors or other chemical stimuli at a later stage of theformation of neo-cartilage, since the cells are washed free of theenzymes before culturing.

b) Expansion of Chondrocytes

The isolated chondrocytes are then expanded by any method suitable forsuch purposes such as, for example, by incubation in a suitable growthmedium, for a period of several days, typically from about 3 to about 30days, preferably for 14 days, at about 37° C. Any kind of culture orincubation apparatus or chamber may be used for expanding chondrocytes.The expansion of the cells is preferably associated with the removal ofdead chondrocytes, residual native extracellular matrix and othercellular debris before the chondrocytes are selected for culturing andmultiplying. Selected chondrocytes are collected and isolated usingtrypsinization process or any other suitable method.

Expanded chondrocytes are then suspended in a suitable solution andseeded into a support matrix to form a seeded matrix. The seeded matrixis typically processed in a tissue processor.

c) Suspension and Seeding of Chondrocytes in the Support Matrix

Following the expansion, chondrocytes are suspended in any suitablesolution, preferably collagen containing solution. For the purposes ofthis invention such solution is typically a gel, preferably sol-geltransitional solution which changes the state of the solution fromliquid sol to solid gel above room temperature. The most preferred suchsolution is the thermo-reversible gelation hydrogel or athermo-reversible polymer gel. The thermo-reversible property isimportant both for immobilization of the chondrocytes within the supportmatrix and for implanting of the neo-cartilage construct within thecartilage lesion.

One characteristic of the sol-gel is its ability to be cured ortransitioned from a liquid into a solid form. This property may beadvantageously used for solidifying the suspension of chondrocyteswithing the support matrix for delivery, storing or preservationpurposes. Additionally, these properties of sol-gel also permit its useas a support matrix by changing its sol-gel transition by increasing ordecreasing temperature, as described in greater detail below forthermo-reversible gelation hydrogel, or exposing the sol-gel to variouschemical or physical conditions or ultraviolet radiation.

In one embodiment the expanded chondrocytes are suspended in acollagenous sol-gel solution before incorporation (seeding) into thesupport matrix. The sol-gel viscosity permits easy mixing ofchondrocytes avoiding need to use shear forces. One example of thesuitable sol-gel solution is the solution substantially composed of TypeI collagen, commercially available under trade name VITROGEN® fromCohesion Corporation, Palo Alto, Calif. VITROGEN is a purifiedpepsin-solubilized bovine collagen dissolved in 0.012N HCl. Sterilecollagen for tissue culture may be additionally obtained from othersources, such as, for example, Collaborative Biomedical, Bedford, Mass.,and Gattefosse, SA, St Priest, France.

When using a VITROGEN solution, the cell density is approximately5-10×10⁶ cells/mL. However, both the density of the cells, the volumefor their seeding and strength of the solution are variables within thealgorithm, and the higher or lower number of chondrocytes may besuspended in a larger or lower volume of the suspension solution,depending on the size of the support matrix and the size of thecartilage lesion.

Seeding of the suspended chondrocytes into the support matrix is by anymeans which permit even distribution of the chondrocytes within saidsupport matrix. Seeding may be achieved by bringing the suspension andthe support matrix into close contact and seeding the cells by wickingor suction of the suspension into the matrix by capillary action, byinserting the support matrix into the suspension, by using suction,positive or negative pressure, injection or any other means which willresult in even distribution of the chondrocytes within said supportmatrix.

In alternative embodiment, the chondrocytes are suspended in thethermo-reversible gelation hydrogel or gel polymer at temperaturebetween 5 and 15° C. At that temperature, the hydrogel is at a liquidsol stage and easily permits the chondrocytes to be suspended in thesol. Once the chondrocytes are evenly distributed within the sol, thesol is subjected to higher temperature of about 30-37° C. at whichtemperature, the liquid sol solidifies into solid gel having evenlydistributed chondrocytes within. The gelling time is from about severalminutes to several hours, typically about 1 hour. In such an instance,the solidified gel may itself become and be used as a support matrix orthe suspension in sol state may be loaded into a separate supportmatrix, such as a sponge or honeycomb support matrix.

Other means of generating suspending gels, not necessarilythermo-reversible, are also available and suitable for use. Polyethyleneglycol (PEG) derivatives, in which one PEG chain contains vinyl sulfoneor acrylate end groups, and the other PEG chain contains free thiolgroups will covalently bond to form thio-ether linkages. If one or bothpartner PEG molecules are branched (three- or four-armed), the couplingresults in a network, or gel. If the molecular weight of the PEG chainsis several thousand Daltons (500 to 10,000 Daltons along any linearchain segment), the network will be open, swellable by water, andcompatible with living cells. The coupling reaction can be accomplishedby preparing 5 to 20% (w/v) solutions of each PEG separately in aqueousbuffers or cell culture media. Chondrocytes can be added to thethiol-PEG solution. Just prior to incorporation into the support matrix,the cells plus thiol PEG and the acrylate or vinyl sulfone PEG are mixedand infused into the matrix. Gelation will begin spontaneously in 1 to 5minutes; the rate of gelation can be modulated somewhat by theconcentration of PEG reagent and by pH. The rate of coupling is fasterat pH 7.8 than at pH 6.9. Such gels are not degradable unless additionalester or labile linkages are incorporated into the chain. Such PEGreagents may be purchased from Shearwater Polymers, Huntsville, Ala.,USA; or from SunBio, Korea.

In a second alternative, alginate solutions can be gelled in thepresence of calcium ions. This reaction has been employed for many yearsto suspend cells in gels or micro-capsules. Cells can be mixed with a1-2% (w/v) solution of alginate in culture media devoid of calcium orother divalent ions, and infused into the support matrix. The matrix canthen be immersed in a solution containing calcium chloride, which willdiffuse into the matrix and gel the alginate, trapping and supportingthe cells. Analogous reactions can be accomplished with other polymerswhich bear negatively charged carboxyl groups, such as hyaluronic acid.Viscous solutions of hyaluronic acid can be used to suspend cells andgelled by diffusion of ferric ions.

Suspension loaded into the support matrix or gelled into the solidsupport is processed using the algorithm of the invention. Suchprocessing is performed in a processing apparatus, such as a TESSprocessor.

C. Preparation of Support Matrix

The support matrix for seeding expanded chondrocytes provides astructural support for growth and two or three-dimensional propagationof chondrocytes. Generally, the support matrix is biologicallybiocompatible, hydrophilic and has preferably a neutral charge.

Typically, the support matrix is a two or three-dimensional structuralcomposition, or a composition able to be converted into such structure,containing a plurality of pores dividing the space into a fluidicallyconnected interstitial network. In some embodiments the support matrixis a sponge-like structure or honeycomb-like lattice.

In general, any polymeric material can serve as the support matrix,provided it is biocompatible with tissue and possesses the requiredgeometry. Polymers, natural or synthetic, which can be induced toundergo formation of fibers or coacervates, can then be freeze-dried asaqueous dispersions to form sponges. Typically, such sponges must bestabilized by crosslinking, such as, for example, ionizing radiation.Practical example includes preparation of freeze-dried sponges ofpoly-hydroxyethyl-methacrylate (pHEMA), optionally having additionalmolecules, such as gelatin, entrapped within advantageously. Such typesof sponges can advantageously function as support matrices for thepresent invention. Incorporation of agarose, hyaluronic acid, or otherbio-active polymers can be used to modulate cellular responses. A widerange of polymers may be suitable for the fabrication of support matrixsponges, including agarose, hyaluronic acid, alginic acid, dextrans,polyHEMA, and poly-vinyl alcohol above or in combination.

Typically, the support matrix is prepared from a collagenous gel or gelsolution containing Type I collagen, Type II collagen, Type IV collagen,gelatin, agarose, hyaluronin, cell-contracted collagens containingproteoglycans, glycosaminoglycans or glycoproteins, fibronectin,laminin, bioactive peptide growth factors, cytokines, elastin, fibrin,synthetic polymeric fibers made of poly-acids such as polylactic,polyglycotic or polyamino acids, polycaprolactones, polyamino acids,polypeptide gel, copolymers thereof and combinations thereof.Preferably, the support matrix is a gel solution, most preferablycontaining aqueous Type I collagen or a polymeric, preferablythermo-reversible, gel matrix.

The gel or gel solution used for preparation of the support matrix istypically washed with water and subsequently freeze-dried or lyophilizedto yield a sponge like matrix able to incorporate or wick thechondrocytes suspension withing the matrix. The cellular support matrixof the current invention acts like a sponge when infiltrated with thechondrocyte suspension wherein the cells are evenly distributed.

One important aspect of the support matrix is the pore size of thesupport matrix. Support matrices having different pore sizes permitfaster or slower infiltration of the chondrocytes into said matrix,faster or slower growth and propagation of the cells and, ultimately,the higher or lower density of the cells in the neo-cartilage construct.Such pore size may be adjusted by varying the pH of the gel solution,collagen concentration, lyophilization conditions, etc. Typically, thepore size of the support matrix is from about 50 to about 500μ,preferably the pore size is between 100 and 300μ and most preferablyabout 200μ.

The support matrix may be prepared according to procedures described inExample 3, or by any other procedure, such as, for example, proceduresdescribed in the U.S. Pat. Nos. 6,022,744; 5,206,028; 5,656,492;4,522,753 and 6,080,194 herein incorporated by reference.

One preferred type of support matrix is Type-I collagen support matrixfabricated into a sponge, commercially available from Koken Company,Ltd., Tokyo, Japan, under the trade name Honeycomb Sponge.

An exemplary neo-cartilage support matrix made of collagen and embeddedwith chondrocytes is seen in FIG. 1, wherein FIG. 1A is a schematicdrawing of the sponge made of sol/gel showing the distribution ofchondrocytes within the collagen sponge. FIG. 1B shows a microphotographof the actual neo-cartilage construct (Neo-Cart™) having 4 mm indiameter and thickness of 1.5 mm. The seeding density of this constructis 300,000-375,000 chondrocytes per 25 μl of collagen solutioncorresponding to about 12-15 millions cells/mL. The cell density rangefor seeding is preferably from about 3 to about 60 millions/mL.

a) Honeycomb Cellular Support Matrix

In one embodiment of the invention, the support matrix is ahoneycomb-like lattice matrix providing a cellular support for activatedchondrocytes, herein described as neo-cartilage.

The honeycomb-like matrix supports a growth platform for theneo-cartilage and permits three-dimensional propagation of theneo-cartilage.

The honeycomb-like matrix is fabricated from a polymerous compound, suchas collagen, gelatin, Type I collagen, Type II collagen or any otherpolymer having a desirable properties. In the preferred embodiment, thehoneycomb-like matrix is prepared from a solution comprising Type Icollagen.

The pores of the honeycomb-like matrix are evenly distributed withinsaid matrix to form a sponge-like structure able to taking in and evenlydistributing the neo-cartilage suspended in a viscous solution.

b) Sol-Gel Cellular Support Matrix

In another embodiment, the support matrix is fabricated from sol-gelmaterials wherein said sol-gel materials can be converted from sol togel and vice versa by changing temperature. For these materials thesol-gel transition occurs on the opposite temperature cycle of agar andgelatin gels. Thus, in these materials the sol is converted to a solidgel at a higher temperature. Sol-gel material is a material which is aviscous sol at temperatures of below 15° and a solid gel at temperaturesaround and above 37°. Typically, these materials change their form fromsol to gel by transition at temperatures between about 15° and 37° andare in transitional state at temperatures between 15° C. and 37°. Themost preferred materials are Type I collagen containing gels and athermo-reversible gelation hydrogel (TRGH) which has a rapid gelationpoint.

In one embodiment, the sol-gel material is substantially composed ofType I collagen and, in the form of 99.9% pure pepsin-solubilized bovinedermal collagen dissolved in 0.012N HCl, is commercially available underthe tradename VITROGEN® from Cohesion Corporation, Palo Alto, Calif. Oneimportant characteristic of this sol-gel is its ability to be cured bytransition into a solid gel form wherein said gel cannot be mixed orpoured or otherwise disturbed thereby forming a solid structurecontaining immobilized chondrocytes.

Type I collagen sol-gel is generally suitable for suspending thechondrocytes and for seeding them into a separately prepared supportmatrix in the sol form and gel the sol into the solid gel by heating thesupport matrix to a proper temperature, usually around 30-37° and, inthis form, processing the embedded support matrix. This type of sol-gelcan also be used as a support matrix for purposes of processing the gelcontaining chondrocytes in the processor of the invention into aneo-cartilage construct.

In another embodiment, the sol-gel is thermo-reversible gelationhydrogel (TRGH). Sol-gel thermo-reversible material for preparation ofsol-gel support matrix is a material which is a viscous sol attemperatures of below 15-30° C. and solid gel at temperatures above30-37° C. The primary characteristic of the thermo-reversible gelationhydrogel (TRGH) is that it gels at body temperature and sols at lowerthan 15-30° C. temperature, that upon its degradation within the body itdoes not leave biologically deleterious material and that it does notabsorb water at gel temperatures. TRGH has a very quick sol-geltransformation which requires no cure time and occurs simply as afunction of temperature without hysteresis. The sol-gel transitiontemperature can be set at any temperature in the range from 5° C. to 70°C. by the molecular design of the thermo-reversible gelation polymer(TGP), a high molecular weight polymer of which less than 5 wt % isenough for hydrogel formation.

The typical TRGH is generally made of blocks of high molecular weightpolymer comprising numerous hydrophobic domains cross-linked withhydrophilic polymer blocks. TRGH has low osmotic pressure and is verystable as it is not dissolved in water when the temperature ismaintained above the sol-gel transition temperature. Hydrophilic polymerblocks in the hydrogel prevent macroscopic phase separation andseparation of water from hydrogel during gelation. These properties makeit especially suitable for safe storing and extended shelf-life.

The thermo-reversible gelation hydrogel (TRGH), particularly aspace-holding thermo-reversible gel (SHTG), should be a compressivelystrong and stable at 37° C. and below till about 32° C., that is toabout temperature of the synovial capsule of the joint which istypically below 37° C., but should easily solubilize below 30-31° C. tobe able to be conveniently removed from the cavity as the sol. Thecompressive strength of the SHTG or TRGH must be able to resistcompression by the normal activity of the joint.

In this regard, the thermo-reversible hydrogel is an aqueous solution ofthermo-reversible gelation polymer (TGP) which turns into hydrogel uponheating and liquefies upon cooling. TGP is a block copolymer composed oftemperature responsive polymer (TRP) block, such aspoly(N-isopropylacrylamide) or polypropylene oxide and of hydrophilicpolymer blocks such as polyethylene oxide.

Thermally reversible hydrogels consisting of co-polymers of polyethyleneoxide and polypropylene oxide are available from BASF Wyandotte ChemicalCorporation under the trade name of Pluronics.

In general, thermo-reversibility is due to the presence of hydrophobicand hydrophilic groups on the same polymer chain, such as in the case ofcollagen and copolymers of polyethylene oxide and polypropylene oxide.When the polymer solution is warmed, hydrophobic interactions causechain association and gelation; when the polymer solution is cooled, thehydrophobic interaction disappears and the polymer chains aredis-associated, leading to dissolution of the gel. Any suitablybiocompatible polymer, natural or synthetic, with such characteristicswill exhibit the same reversible gelling behavior.

This type of thermo-reversible gelation hydrogel is particularlypreferred for preparation of neo-cartilage constructs for implantationof the construct into the lesion. In such an instance, the harvestedchondrocytes are suspended in the TRGH sol, then warmed to about 37° C.into the solid gel which thus itself becomes a seeded support matrix,then submitting said seeded matrix to the processing in the tissueprocessor using the algorithm of the invention, including resting periodas described below, thereby resulting in a formation of theneo-cartilage construct, then submitting said construct to cooling tochange its form into a sol and in this form injecting the neo-cartilageinto the lesion wherein upon warming to body temperature the sol isimmediately converted into the gel containing neo-cartilage. In time,the delivered neo-cartilage is integrated into the existing cartilageand the TRGH is subsequently degraded leaving no undesirable debrisbehind.

D. Processing Neo-Cartilage and Tissue Processors

In order to promote three-dimensional growth and propagation ofchondrocytes and/or neo-cartilage, it is advantageous and/or necessaryin certain instances to facilitates such growth and propagation bychanging conditions of their growth. Such facilitation may be initiatedeither ex vivo, in vitro or in vivo.

This process is, in the current invention, achieved by subjecting eitherthe suspended expanded chondrocytes or the support matrix incorporatedwith suspended chondrocytes to certain protocol (the algorithm) ofconditions which were found to promote such propagation. Such conditionsare, for example, application of constant or cyclic hydrostaticpressure, resting periods at static pressure, recirculation and changingflow rate of media, regulation of oxygen or carbon dioxideconcentrations, cell density, control pH, availability of nutrients andco-factors, etc. Typically, this process is performed in the apparatus,preferably in the TESS™ tissue processor, permitting changing of theconditions, as stated above.

a) Neo-Cartilage Tissue Processor

The general design of the tissue processor is the apparatus forculturing chondrocytes comprising a culture unit having a culturechamber containing culture medium and a supply unit for the continuousand intermittent delivery of the culture medium, a pressure generatorfor applying atmospheric or constant or cyclic hydrostatic pressureabove the atmospheric pressure to chondrocytes in the tissue chamber,said generator having means for changing the pressure, timing, orapplying the atmospheric, constant or cyclic hydrostatic pressure atpredetermined periods and, optionally, a means capable of deliveringand/or absorbing gases such as nitrogen, carbon dioxide and oxygen.Additionally, the processor typically comprises a hermetically sealedspace including a heating, cooling and humidifying means.

An exemplary scheme of the tissue processor suitable for applying ofstatic or hydrostatic pressure, changing flow rate of the medium andregulating gas concentration delivered to the embedded support systemsuitable for purposes of this invention is seen in FIG. 2A. The tissueprocessor, seen in FIG. 2B, known as Tissue Engineering Support System(TESS) is described in the U.S. Pat. No. 6,432,713 issued on Aug. 13,2002, and also in the U.S. application Ser. No. 09/895,162, both herebyincorporated by reference.

b) Biochemical and Histological Testing of Neo-Cartilage Constructs

The neo-cartilage constructs are tested for their metabolic activity,genetic activation and histological appearance.

Typically, the constructs are harvested at days 6 and 18. Forhistological evaluation of the immature and mature cartilage matrix, 4%paraformaldehyde-fixed paraffin sections are stained with Safranin-O andType II collagen antibody. For biochemical analysis, neo-cartilageconstructs are digested in papain at 60° C. for 18 hours and DNA ismeasured using, for example, Hoechst 33258 dye method as described inAnal. Biochem., 174:168-176 (1988). The production of glycoaminoglycan(GAG) or sulfated-glycosaminoglycan (S-GAG) indicating a metabolicactivity of the chondrocyte culture is tested using, for example,modified dimethylene blue (DMB) microassay according to ConnectiveTissue Research, 9:247-248 (1982).

c) Conditions for Propagation of Chondrocytes, Preparation ofNeo-Cartilage and Neo-Cartilage Constructs

Neo-cartilage construct, as used herein, means a matrix embedded withchondrocytes and processed according to the invention.

Neo-cartilage constructs may be produced as 3-dimensional patchescomprising neo-cartilage having an approximate size of the lesion intowhich they are deposited or they may be produced as 3-dimensional sheetfor use in repairs of extensive cartilage injuries. Their size and shapeis determined by the shape and size of the support matrix. Theirfunctionality is determined by the conditions (the algorithm) underwhich they were processed.

Conditions for three-dimensional propagation of chondrocytes in thesupport matrix into neo-cartilage construct are variable and areadjusted according to the intended use and/or function of theneo-cartilage and depend on the type of used thermo-reversible hydrogeland on the density of the seeded cells. Thus for production of smallneo-cartilage constructs, the conditions will be different from thoseneeded for production of large constructs or for production of extensiveneo-cartilage sheets for partial or total replacement of extensivelydamaged or diseased, for example osteoarthritic, cartilage.

i) Processing Neo-Cartilage Under Variable Flow

One aspect of this invention is the discovery that if the support matrixseeded with chondrocytes is perfused under varying medium flow rates,the cell proliferation, measured by increased accumulation of theextracellular matrix, can be advantageously increased or decreased.Generally, the lower medium flow rate results in the higherextracellular matrix accumulation.

Perfusion is an important variable condition for culturing chondrocytesincorporated into support matrices. Using a faster perfusion flow ratemay slow down extracellular matrix accumulation affecting growth andpropagation of chondrocytes, as measured by production of sulfatedglycosaminoglycan (S-GAG). A slower perfusion rate, on the other hand,results in higher production of S-GAG. These results are important forcontrolling the neo-cartilage growth and for, for example, storage,preservation, transport and shelf-life of neo-cartilage constructs.

The perfusion flow rate suitable for purposes of this invention is fromabout 1 to about 500 μl/min, preferably from about of 5 to about 50μl/min. At the medium perfusion rate 5 μl/min the accumulation ofextracellular matrix is significantly (p<0.05) increased compared toaccumulation of extracellular matrix observed following perfusion atrate 5 μl/min. The optimum flow rate depends upon the total number ofcells in the culture chamber.

ii) Processing Neo-Cartilage Under Different Types of Pressure

Subjecting the seeded support matrix to hydrostatic pressure, inconjunction with a decreased perfusion flow, is an integral part of theculture processing system according to this invention. Different typesof hydrostatic pressure have a significant effect on glycosaminoglycanproduction and thus on extracellular matrix accumulation compared to theeffect of atmospheric pressure alone. The hydrostatic pressure,particularly cyclic hydrostatic pressure applied according to thisinvention has been found to stimulate chondrocyte proliferation andmetabolism which contributes to extracellular matrix accumulation.

Hydrostatic pressure suitable for processing chondrocytes embeddedwithin the support matrix is either a constant or cyclic hydrostaticpressure, such pressure being the pressure above the atmosphericpressure. The cyclic hydrostatic pressure suitable for use in processingof the seeded support matrix is from about 0.01 to about 10.0 MPa,preferably from about 0.5 to about 5.0 MPa and most preferably at about3.0 MPa at 0.01 Hz to about 2.0 Hz, preferably at about 0.5 Hz, appliedfor about 1 hour to about 30 days, preferably about 7 to about 14 days,with or without resting period. Typically, the period of hydrostaticpressure is followed by the resting period, typically from about 1 dayto about 60 days, preferably for about 7 to about 28 days, mostpreferably for about 12 to about 18 days.

Studies performed in support of this invention indicate that cellviability is not affected by the hydrostatic pressure and is maintainedwith chondrocytes distributed uniformLy within the support matrix.Following the treatment with hydrostatic pressure, accumulations of bothDNA and S-GAG are significantly increased compared to cultures notexperiencing applied load, indicating that chondrocyte activation andmetabolic and genetic activity can be controlled by the cultureenvironment.

iii) Processing Neo-Cartilage Under Reduced Oxygen Concentration

Another variable in the processing of seeded support matrices is theconcentration of oxygen, carbon dioxide and nitrogen.

The chondrocytes-embedded support matrix described above may be furthercultured under reduced O₂ concentration (i.e. less than 20% saturation)during formation of neo-cartilage in the TESS processor. The reducedoxygen concentration of cartilage has been observed in vivo, and suchreduction may be due to its normal lack of vascularization whichproduces a lower oxygen partial pressure, as compared to the adjacenttissues. In this set of studies, chondrocytes seeded in support matrixor neo-cartilage were cultured under oxygen concentration between about0% and about 20% saturation or under dioxide concentration about 5%.

E) Determination of Conditions for Optimization of the Algorithm

The ultimate aim of this invention was to find and confirm conditions(the algorithm) for preparation of neo-cartilage constructs forimplantation into cartilage lesions, which in conjunction withdeposition of one or two sealant layers, would lead to healing of thedamaged, injured, diseased or aged cartilage by a) growth of superficialcartilage layer completely overgrowing and covering the lesion andprotecting implanted neo-cartilage construct; b) integration ofneo-cartilage implanted into the lesion as the neo-cartilage construct;and c) subsequent degradation of the construct and sealant materials.

The underlying studies, described below, show that a properly designedand optimized culture conditions utilizing hydrostatic pressure withmedium perfusion followed by constant culture result in fabrication ofneo-cartilage constructs which are integrated into the native cartilagewhen implanted under the one layer or in between two layers of sealantsaccording to the invention.

General design for a method for preparation of neo-cartilage constructscomprises steps:

-   -   a) isolation of chondrocytes from a donor tissue;    -   b) expanding the chondrocytes for about 3-28 days;    -   c) seeding chondrocytes in a thermo-reversible or collagen gel        or collagen sponge support matrix;    -   d) subjecting the seeded gel or sponge to a static, constant or        cyclic hydrostatic pressure above atmospheric pressure (about        0.5-3.0 MPa at 0.5 Hz) with medium perfusion rate of 5 μl/min        for several (5-10) days; and    -   e) subjecting the seeded gel or sponge to resting period for ten        to fourteen days at constant (atmospheric) pressure.

Neo-cartilage constructs obtained by the above-outlined conditions andmethod show that the combined algorithm of hydrostatic pressure andstatic pressure has advantage over conventional culture methods byresulting in higher cell proliferation and extracellular matrixaccumulation. Use of thermo-reversible or collagen gel or collagensponge support matrix maintains uniform cell distribution within thesupport matrix and also provides support for newly synthesizedextracellular matrix. Obtained 3-dimensional neo-cartilage construct iseasy to handle and manipulate and can be easily and safely implanted ina surgical setting.

Combination of a period of cyclic hydrostatic pressure under low mediumperfusion rate followed up with a period of static culture (restingperiod) results in increased cell proliferation, increased production ofType II collagen, increased DNA content and increased S-GAGaccumulation.

Increased cell proliferation shows that the harvested inactivenon-dividing chondrocytes have been activated into neo-cartilagecontaining active, dividing and multiplying chondrocytes. Increasedlevel of DNA shows genetic activation of inactive chondrocytes.Increased production of Type II collagen and S-GAG shows that productionof the extracellular matrix has been activated using the algorithmdescribed above.

Although the optimized algorithm described above is preferred, it is tobe understood that this algorithm may be advantageously changed usingvariations of ranges of cyclic hydrostatic pressure, flow rate, durationof the pressure and resting period as disclosed above in detaildescription of each condition. All variations of all conditions andcombinations thereof are intended to be within the scope of thisinvention.

F. Supporting Experimental Studies

In order to test effects of different conditions on the propagation ofchondrocytes within the support matrix for fabrication of theneo-cartilage construct, studies combining conditions described abovefor process optimization were performed during development of thisinvention. Results are shown in FIGS. 3-9 and in Tables 1-3.

For all following studies, the experimental design was as follows withchanges in studies conditions.

Cartilage was harvested under sterile conditions from the trachea of theswine hind limbs, minced and digested, as described in Example 7.Chondrocytes were expanded for 5 days at 37° C. and suspended inVITROGEN® (300,000/30 μl). The suspension was absorbed into a supportmatrix, usually a collagen sponge (4 mm in diameter and 2 mm inthickness) as seen in FIG. 1, commercially available from Koken Co.,Tokyo, Japan. The sponges seeded with chondrocytes were pre-incubatedfor 1 hour at 37° C. to gel the collagen, followed by incubation inculture medium at 37° C., 5% CO₂ and cultured in the Tissue EngineeringSupport System (TESS™) processor seen in FIG. 2.

a) Evaluation of Effect of Hydrostatic Pressure

To evaluate the effect of the pressure and/or medium perfusion rate, thecell seeded sponges were subjected to medium perfusion at 5 μl/min(0.005 mL/min) or 50 μl/min (0.05 mL/min) under the cyclic (Cy-HP) orconstant hydrostatic pressure (constant-HP) of 0.5 MPa at 0.5 Hz for 6days in the TESS processor. Resting period under atmospheric pressurefollowed for 12 days. Some seeded sponges served as controls. These wereincubated under the atmospheric pressure and without perfusion at 37° C.for a total of 18 days in culture. Sponges harvested 24 hours afterseeding with cells (day 0) served as an initial control. More detailedconditions are to be found in Examples and in the following text.

At the end of culture period, the support matrices were harvested forbiochemical and histological analysis. Sulfated glycosaminoglycanproduction was measured using a modified dimethylmethylene bluemicroassay. Histological analysis utilized Safranin-O staining. Moredetailed conditions are to be found in Examples.

The first study was directed to determination of effect of constant(atmospheric), cyclic or constant hydrostatic pressure on production ofS-GAG.

At the end of the culture period, both control and test matrices wereharvested for biochemical and histological analysis. For biochemicalanalysis, production of sulfated glycosaminoglycan (S-GAG μg/cellconstruct) was measured using a modified dimethylmethylene blue (DMB)and DNA microassays described in Example 7. Results are seen in Tables 1and 2 and FIGS. 3-6.

Results of some studies are seen in Tables 1 and 2 showing a numericalrepresentation of observed increase in S-GAG production in matricestreated with the algorithm of the invention. TABLE 1 Pressure ConditionsIn TESS (3 MPa Cyclic In Incubator Total S-GAG Production GroupPressure, (Atmospheric days in (μg/cell construct) (n = 6) @0.5 Hz)Pressure) Culture (Mean ± SD) Initial —  0 day 0 12.56 ± 0.99 Control —18 days 18 57.73 ± 6.43 Test 6 days 12 days 18 *76.32 ± 4.12 (*p < 0.05, compared to Control)

Table 1 summarizes results obtained from seeded matrices (n=6) subjectedeither to atmospheric pressure in an incubator for 18 days (control) orto processing in TESS processor under 3 MPa cyclic hydrostatic pressureat 0.5 Hz for 6 days, followed by 12 days in incubator at atmosphericpressure (test).

As seen in Table 1, S-GAG production (μg/cell construct) per seededmatrix was significantly increased to 132% for test compared to 100%control (FIG. 3A). Histological results seen in FIGS. 3B and 3C(Safranin-O staining for S-GAG) were consistent with the results seen inTable 1 obtained biochemically. FIG. 3B is a photomicrograph ofSafranin-O staining for S-GAG on paraffin sections in 18 days subjectedto static pressure. FIG. 3C is a photomicrograph of Safranin-O stainingfor S-GAG on paraffin sections in cell constructs subjected to cyclichydrostatic pressure for 6 days followed by 12 days of static culture.

As seen in FIG. 3B, when the cell constructs are subjected to staticatmospheric pressure (FIG. 3B), there is much lower S-GAG accumulationin the constructs than when it is subjected to a cyclic hydrostaticpressure for 6 days, followed by 12 days of static atmospheric pressure(FIG. 3C).

To determine the effect of the hydrostatic pressure on chondrocyteproliferation stimulation and matrix accumulation, cartilage washarvested under sterile conditions as described above. Chondrocytes wereexpanded for 5 days at 37° C. and suspended in VITROGEN® (300,000/30μl). The suspension was absorbed into a honeycomb support matrix orcollagen sponge as seen in FIG. 1. The cell constructs were incubated inculture medium at 37° C., 5% CO₂ and 20% O₂, at 0.5 MPa cyclichydrostatic pressure or 0.5 MPa constant hydrostatic pressure for 6 daysfollowed by incubation for 12 days at atmospheric pressure in the TissueEngineering Support System (TESS™) processor seen in FIG. 2. Theremaining cell matrices comprising the control group were incubated atatmospheric pressure for 18 days at 37° C., 5% CO₂ and 20% O₂.

At the end of the culture period, the matrices were harvested forbiochemical analysis. Results are seen in Table 2. Glycosaminoglycanproduction was measures using a modified dimethylmethylene blue (DMB)microassay. Cell proliferation was measured using a modified Hoechst DyeDNA assay. Formation of neo-tissue was evaluated by Safranin-O staining.Results are seen in FIGS. 4A, 4B, 5A and 5B and in Table 2. TABLE 2Pressure Conditions S-GAG Days in Total GAG Production In TESS Incubatordays (μg/cell DNA Group Type of Time/ (Atmospheric In construct) DNAIndex (n = 7) Pressur Days Pressure) culture (Mean ± SD) (Control = 1)Control — — 18 18 59.85 ± 7.69 1 Cy-HP 0.5 MPa 6 12 18 *91.05 ± 10.681.49 Cyclic Const-HP 0.5 MPa 6 12 18 *97.85 ± 5.53  1.74 Constant(*p < 0.05, compared to Control)

All cultures were incubated at 37° C., 5% CO₂ and 20% O₂. In TESSculture, the medium flow rate was 50 μl/min. Two cell matrices from eachgroup were harvested for histological analysis.

As seen in Table 2, the matrices subjected to conditions listed in thecontrol group, cyclic hydrostatic pressure (Cy-HP) and constanthydrostatic pressure (const-HP) groups resulted in production of 59.85,91.05 and 97.85 μg/cell construct of S-GAG and 1, 1.49 and 1.74(control=1) of DNA content Index, respectively. These results clearlyshow that neo-cartilage cultured under hydrostatic pressure, whethercyclic or constant, followed by static culture is more genetically andmetabolically active than the neo-cartilage treated under staticatomospheric conditions (controls). These results are graphicallyillustrated in FIG. 4 which shows effect of hydrostatic pressure onproduction of sulfated glycosaminoglycan (FIG. 4A) and DNA content index(FIG. 4B).

FIG. 4A is a graphical representation of results enumerated in Table 2and shows the sulfated glycosaminoglycan production in μg/cell constructwherein control represents seeded matrices subjected to atmosphericpressure, Cy-HP represents seeded matrices subjected to cyclichydrostatic pressure (0.5 MPa) and constant-HP represent matricessubjected to constant hydrostatic pressure (0.5 MPa).

Results seen in Table 2 are illustrated graphically in FIG. 4A, underthe conditions described above. There was significant increase in S-GAGproduction for both the cyclic (Cy-HP) and constant hydrostatic pressure(constant-HP) groups compared to atmospheric pressure (control) group.Specifically, the production of S-GAG in the control group was 59.85μg/cell construct. In the group Cy-HP the production was 91.05 μg/cellconstruct. In the group constant-HP cell construct production was 97.85μg/cell construct resulting in increase of S-GAG production to 152% forgroup Cy-HP and to 162% for the group constant-HP compared to thecontrol group.

FIG. 4B shows DNA production with corresponding results presented inTable 2 for DNA, likewise showing increased production of DNA inconstructs processed under cyclic or constant hydrostatic pressure.

FIG. 5A is a graph comparing effect of constant atmospheric pressure(Control) and zero MPa hydrostatic pressure (0 MPa) serving as pressurecontrols, 0.5 MPa cyclic hydrostatic pressure (Cy-HP) and 0.5 MPaconstant hydrostatic pressure (constant-HP) at day 6 and 18 on supportmatrices subjected to processing in the TESS processor. All matriceswere incubated at 37° C. for 18 days. The Cy-HP and constant-HP wereapplied for the first 6 days followed by 12 days of incubation atatmospheric pressure.

Results seen in FIG. 5A show that combination of Cy-HP or constant-HPwith resting period of atmospheric pressure incubation resulted insignificant (p<0.05) increase of S-GAG production in the processedmatrices compared to S-GAG production observed in matrices processed atatmospheric pressure with perfusion only.

FIG. 5B shows the index of DNA content (Initial=1) in matrices subjectedto static (Control), zero hydrostatic (0 MPa), cyclic (Cy-HP) orconstant (Constant-HP) hydrostatic pressure for 6 day and 12 days ofatmospheric pressure culture. Increase in DNA content in matricessubjected to the algorithm conditions is clearly shown in both cyclicand constant hydrostatic pressure groups. Comparison of the initial andcontrol DNA level to DNA levels in all three groups subjected tohydrostatic pressure reveals that the DNA level in constructs subjectedto the cyclic hydrostatic pressure is higher at day 6 than at day 18 andthe DNA level in constructs subjected to constant hydrostatic pressureis lower at day 6 than at day 18. Highest levels of DNA is observed inmatrices submitted to constant hydrostatic pressure at day 18.

FIGS. 6A and 6B show histological evaluation of matrices by Safranin-O.FIG. 6A shows accumulation of S-GAG on day 18 in matrices subjected toatmospheric pressure. FIG. 6B shows accumulation of S-GAG in matricessubjected to 6 days of cyclic hydrostatic pressure (Cy-HP), followed by12 days of atmospheric pressure. The greater S-GAG accumulation in Cy-HPculture matrices is evident from the increased density of thephotomicrograph clearly visible in the construct. FIG. 3C showsaccumulation of Type II collagen in matrices subjected to theatmospheric pressure or to the cyclic hydrostatic pressure (FIG. 6D).Larger accumulation of Type II collagen in FIG. D is clearly seen.

These results demonstrate that chondrocytes may be placed in culture tocoalesce into a neo-cartilage construct with accumulated extracellularmatrix macro molecules, such as sulfated glycosaminoglycan (S-GAG).

b) Evaluation of Effect of Perfusion Flow

The second type of study was performed in order to determine the effectof perfusion flow rate on chondrocyte proliferation (DNA content) andproduction of extracellular matrix (S-GAG accumulation). Results areseen in Table 3 and FIGS. 7A and 7B.

FIG. 7 describes results of studies of the effect of the perfusion flowrate on cell proliferation measured by levels of DNA content index (FIG.7A) and, S-GAG accumulation (FIG. 7B) at day 0, 6 and 18.

FIG. 7A shows that the lower perfusion rate (5 μl/min) results in higherDNA content index used as a measure for determination of cellproliferation. Specifically, the DNA content index compared to theinitial DNA content index equal to 1 increased by about 50% to about 1.5when the culture perfusion rate was 5 μl/min. The higher perfusion rate(50 μl/min) resulted in much smaller increase in DNA content index toabout 1.2.

Table 3 shows the effect of perfusion flow rate on the S-GAG productionin matrices treated as outlined above where the flow rate was either0.05 mL/min (50 μl/min) or 0.005 mL/min (5 μl/min). TABLE 3 Cultureduration In TESS In GAG Medium (0.5 Incubator Total Production PerfusionMPa (Atmos- days (μg/cell Group Flow Rate Cyclic pheric in construct) (n= 7) (mL/min) Pressure Pressure) culture (Mean ± SD) A  0.05 mL/min 6days 12 days 18 days  78.75 ± 6.84 B 0.005 mL/min 6 days 12 days 18 days107.33 ± 8.53

All cultures were incubated at 37° C., 5% CO₂ and 20% O₂. In theculture, 0.5 MPa cyclic pressure at 0.5 Hz was applied to the cellmatrices. Two matrices from each group were harvested for histologicalanalysis.

As seen in Table 3, the lower perfusion rate (5 μl/min) resulted inapproximately 1.5 higher production of S-GAG than the higher perfusionrate (50 μl/min).

These results are seen in graphical form in FIG. 7B. FIG. 7B is graphshowing differences between S-GAG production by seeded support matricessubjected to a medium perfusion flow rate of 5 μl/min compared tomatrices subjected to a medium perfusion flow rate of 50 μl/min at days6 and 18. As seen in FIG. 7B, increase in S-GAG production up to 136%(p<0.05) in matrices subjected to a slower rate of 5 μl/min.

The results summarized in FIGS. 7A and 7B clearly show a significantincrease in both the DNA content index and S-GAG production in the cellconstruct at a flow rate of 5 μl/min compared to the flow rate 50 μl/mL.There is no significant difference in the amount of S-GAG released intothe medium between the two flow rates. It is therefore possible to uselower flow rate and avoid shear.

Determination whether the combination of the perfusion flow rate withcyclic or constant hydrostatic pressure leads to increased formation ofextracelluar matter was also studied. Results are seen in FIG. 8.

FIG. 8 illustrates a formation of extracellular matrix after 15 daysculture determined in matrices treated with perfusion (5 μl/min) only(FIG. 8A), cyclic hydrostatic pressure 2.8 MPa at 0.015 Hz (FIG. 8B) andconstant hydrostatic pressure 2.8 MPa at 0.015 Hz (FIG. 8C) asdetermined by toluidine blue staining. This figure clearly shows thathydrostatic pressure and medium perfusion enhances production ofextracellular matrix.

C. Evaluation of Effect of Low Oxygen Tension

The third type of study was performed in order to determine the effectof low oxygen tension on chondrocyte proliferation (DNA content) andproduction of extracellular matrix (S-GAG accumulation). Results areseen in Table 4 and FIGS. 9A and 9B. TABLE 4 Culture duration In OxygenIn TESS Incubator Total GAG Production con- (0.5 MPa (Atmos- days(μg/cell Group centration Cyclic pheric in construct) (n = 8) (%)Pressure) Pressure) culture (Mean ± SD) A 20% 7 days 14 days 21 days 60.89 ± 6.02 B  2% 7 days 14 days 21 days *105.59 ± 10.95(*p < 0.05, compared to group A)

All cultures were incubated at 37° C., at 5% CO₂. In TESS culture, themedium flow rate was 5 μl/min. Two cell matrices from each group wereharvested for histological analysis.

As seen in Table 4, the lower oxygen tension (2% O₂ concentration)resulted in approximately 1.7 higher production of S-GAG than higheroxygen concentration (20%) corresponding to atmospheric O₂concentration. These results are seen in graphical form in FIG. 9A.

FIG. 9A is a graph showing differences between S-GAG production by cellconstructs subjected to 2% oxygen concentration (Cy-HP) and to cyclichydrostatic pressure followed by static pressure compared to cellconstructs subjected to 20% oxygen concentration and Cy-HP followed bystatic pressure. As already seen in Table 4, at 2% oxygen concentrationcompared to 20% concentration, the production of S-GAG rose byapproximately 70%.

FIG. 9B shows the DNA content index (initial=1) in cell constructssubjected to 2% or 20% oxygen concentration and Cy-HP pressure followedby static pressure. There are no significant differences in the DNAcontent index between 2% oxygen concentration and 20% oxygenconcentration. These results indicate that the lower oxygen tensionstimulates S-GAG production in cell constructs when combined with thecyclic hydrostatic culture followed by static culture. However, the cellproliferation, expressed as DNA content index, is not affected bychanges in oxygen tension.

The algorithm of the invention thus comprises at least a combination ofthe low perfusion flow rate from about 1 to 500 μL/minute, preferablyabout 5 to 50 μL/minute, most preferably about 5 μL/minute, low oxygenconcentration from about 1% to about 20%, preferably about 2% to about5%, with a certain predetermined period of cyclic or constanthydrostatic pressure from zero to about 10 MPa at about 0.01 to about 1Hz, preferably about 0.1 to about 0.5 Hz, from about zero to about 10MPa of cyclic or constant hydrostatic pressure, preferably about 0.05MPa to about 3 MPa at about 0.1 to about 0.5 Hz, followed by the periodof a static atmospheric pressure. The algorithm conditions are appliedfrom about 1 hour to about 90 days wherein the time for applying thehydrostatic pressure is from zero to about 24 hours per day for fromabout one day to about ninety days, wherein said hydrostatic pressure ispreceded or followed by a period of zero to about 24 hours of a staticatmospheric pressure for from about one day to about ninety days withpreferred time for applying the hydrostatic cyclic or constant pressureof about 7 to 28 days followed or preceded by a period of zero to about28 days of the atmospheric pressure.

d) General Applicability of the Algorithm of the Invention to VariousCell Types

The algorithm described above for chondrocytes is similarly applicableto other types of cell and tissue, such as fibroblasts,fibrochondrocytes, tenocytes, osteoblasts and stem cells capable ofdifferentiation, or tissues such as cartilage connective tissue,fibrocartilage, tendon and bone. The algorithm conditions may be thesame or different but would be generally within the above describedranges.

II. Neo-Cartilage Composition Construct

The neo-cartilage composition construct is a multilayeredthree-dimensional structure comprised at least of living chondrocytesincorporated into a cellular support matrix. The support matrix isembedded with living chondrocytes.

The construct is fabricated in vitro and ex vivo prior to implantinginto the cartilage lesion. The construct is fabricated using the methodand conditions, cumulatively called the algorithm, described above, withall conditions being variable within the given ranges and depending onthe intended use or on the method of delivery.

In one embodiment, the autologous or heterologous chondrocytes arecultured as described, embedded into the support matrix and processedinto the neo-cartilage construct using predetermined medium perfusionflow rate, cyclic or constant hydrostatic pressure and reduced orincreased concentration of oxygen and/or carbon dioxide. Theneo-cartilage construct is delivered into the cartilage lesion cavityand deposited between two layers of sealant and left in situ to beintegrated into the native cartilage.

III. Method for Formation of Superficial Cartilage Layer

The primary aspect of this invention is a finding that when theneo-cartilage, neo-cartilage construct or seeded support matrix producedaccording to procedures and conditions described above is implanted intoa cartilage lesion cavity and covered with a biocompatible adhesivesealant, the resulting combination leads to a formation of a superficialcartilage layer completely overgrowing said lesion.

The method is based on producing a neo-cartilage and neo-cartilageconstruct comprising support matrix seeded with expanded chondrocytesprocessed according to the algorithm of the invention. Chondrocytes aretypically suspended in a collagen sol which is thermo-reversible andeasily changes from sol to gel at the body temperature therebypermitting external preparation of and delivery of the neo-cartilageconstruct into the lesion in form of the sol which changes its stateinto gel upon delivery to the lesion and warming to the bodytemperature.

The neo-cartilage construct is implanted into the lesion and covered bya layer of a biologically acceptable adhesive sealant. Optionally, thefirst layer of the sealant is introduced into the lesion and depositedat the bottom of the lesion. This first sealant's function is to prevententry and to block the migration of subchondral and synovial cells ofthe extraneous components, such as blood-borne agents, cell and celldebris, etc., into the cavity and their interference with theintegration of the neo-cartilage therein. The second sealant layer isplaced over the surface of the construct. The presence of both thesesealants in combination with the neo-cartilage construct results insuccessful integration of the neo-cartilage into the joint cartilage.

The method may be practiced in several modes and each mode involvesgeneric steps outlined below in variable combinations.

General way to practice the method for repair and restoration ofdamaged, injured, diseased or aged cartilage to a functional cartilageis to follow steps:

a) Preparing Neo-Cartilage. Neo-Cartilage Construct or ChondrocyteSupport Matrix

This step involves preparation of neo-cartilage, neo-cartilagecomprising constructs and support matrix comprising autologous orheterologous chondrocytes incorporated therein. Preparation of any ofthe three entities named above is described in greater detail above insections I.B-D.

b) Depositing the First and Second Sealant into the Lesion

This step involves introducing a first and a second layer of a first anda second biologically acceptable sealant into a cartilage lesion. Thefirst and second sealants may be the same or different. It is to beunderstood that the utilization of the first bottom layer is optionaland that the method for a formation of the superficial cartilage layeris enabled without the first layer.

Specifically, this step involves deposition of the first sealant at thebottom of the lesion and of the second sealant over the lesion. Thefirst and the second sealants can be the same or different, however,both the first and the second sealants must have certain definiteproperties to fulfill their functions.

The first sealant, deposited into the cavity before the neo-cartilage isdeposited, acts as a protector of the lesion cavity integrity, that is,it protects the lesion cavity not only from extraneous substances but italso protect this cavity from formation of the fibrocartilage in theinterim when the cavity is filled with a space-holding gel inexpectation of implantation of the neo-cartilage after processing. Thesecond sealant acts as a protector of the lesion cavity on the outsideas well as a protector of the neo-cartilage construct deposited within acavity formed between the two sealants and as well as an initiator ofthe formation of the superficial cartilage layer.

1. First Sealant

The optionally deposited first sealant forms an interface between theintroduced neo-cartilage construct and the native cartilage. The firstsealant, deposited at the bottom of the lesion, must be able to protectthe construct from and prevent chondrocyte migration into thesub-chondral space. Additionally, the first sealant prevents theinfiltration of blood vessels and undesirable cells and cell debris intothe neo-cartilage construct and it also prevents formation of thefibrocartilage.

2. Second Sealant

The second sealant acts as a protector of the neo-cartilage construct orthe lesion cavity on the outside and is typically deposited over thelesion either before or after the neo-cartilage is deposited therein andin this way protects the integrity of the lesion cavity from anyundesirable effects of the outside environment, such as invading cellsor degradative agents and seals the space holding gel in place beforethe neo-cartilage is deposited therein. The second sealant also acts asa protector of the neo-cartilage construct implanted within a cavityformed between the two sealants. In this way, the second sealant may bedeposited after the neo-cartilage is implanted over the first sealantand seal the neo-cartilage within the cavity or it may be deposited overthe space holding gel. The third function of the second sealant is as aninitiator or substrate for the formation of a superficial cartilagelayer. Studies performed during the development of this inventiondiscovered that when the second sealant was deposited over the cartilagelesion, a growth of the superficial cartilage layer occurred as anextension of the native superficial cartilage layer. This superficialcartilage layer is particularly well-developed when the lesion cavity isfilled with the space-holding or thermo-reversible gel thereby leadingto the conclusion that such a gel might provide a substrate for theformation of such superficial cartilage layer.

3. First and Second Sealant Properties

The first or second sealant of the invention must possess the followingcharacteristics:

Sealant must be biologically acceptable, easy to use and possessrequired adhesive and cohesive properties.

The sealant is biologically compatible with tissue, be non-toxic, notswell excessively, not be extremely rigid or hard, as this could causeabrasion of or extrusion of the sealant from the tissue site, must notinterfere with the formation of new cartilage, or promote the formationof other interfering or undesired tissue, such as bone or blood vesselsand must resorb and degrade by an acceptable pathway or be incorporatedinto the tissue.

The sealant must rapidly gel from a flowable liquid or paste to aload-bearing gel within 3 to 15 minutes, preferably within 3-5 min.Longer gelation times are not compatible with surgical time constraints.Additionally, the overall mode of use should be relatively simplebecause complex procedures will not be accepted by surgeons.

Adhesive bonding is required to attach the sealant formulation to tissueand to seal and support such tissue. Minimal possessing peel strengthsof the sealant should be at least 3 N/m and preferably 10 to 30 N/m.Additionally, the sealant must itself be sufficiently strong so that itdoes not break or tear internally, i.e., it must possess sufficientcohesive strength, measured as tensile strength in the range of 0.2 MPa,but preferably 0.8 to 1.0 MPa. Alternatively, a lap shear measurementmay be given to define the bond strength of the formulation should havevalues of at least 0.5 N/cm² and preferably 1 to 6 N/cm².

Sealants possessing the required characteristics are typicallypolymeric. In the un-cured, or liquid state, such sealant materialsconsist of freely flowable polymer chains which are not cross-linkedtogether, but are neat liquids or are dissolved in physiologicallycompatible aqueous buffers. The polymeric chains also possess sidechains or available groups which can, upon the appropriate triggeringstep, react with each other to couple, or cross-link the polymer chainstogether. If the polymer chains are branched, i.e., comprising three ormore arms on at least one partner, the coupling reaction leads to theformation of a network which is infinite in molecular weight, i.e., agel.

The formed gel has cohesive strength dependent on the number ofinter-chain linkages, the length (molecular weight) of the chainsbetween links, the degree of inclusion of solvent in the gel, thepresence of reinforcing agents, and other factors. Typically, networksin which the molecular weight of chain segments between junction points(cross-link bonds) is 100-500 Daltons are tough, strong, and do notswell appreciably. Networks in which the chain segments are 500-2500Daltons swell dramatically in aqueous solvents and become mechanicallyweak. In some cases the latter gels can be strengthened by specificreinforcer molecules; for example, the methylated collagen reinforcesthe gels formed from 4-armed PEGs of 10,000 Daltons (2500 Daltons perchain segment).

The gel's adhesive strength permits bonding to adjacent biologicaltissue by one or more mechanisms, including electrostatic, hydrophobic,or covalent bonding. Adhesion can also occur through mechanicalinter-lock, in which the uncured liquid flows into tissue irregularitiesand fissures, then, upon solidification, the gel is mechanicallyattached to the tissue surface.

At the time of use, some type of triggering action is required. Forexample, it can be the mixing of two reactive partners, it can be theaddition of a reagent to raise the pH, or it can be the application ofheat or light energy.

Once the sealant is in place, it must be non-toxic to adjacent tissue,and it must be incorporated into the tissue and retained permanently, orremoved, usually by hydrolytic or enzymatic degradation. Degradation canoccur internally in the polymer chains, or by degradation of chainlinkages, followed by diffusion and removal of polymer fragmentsdissolved in physiological fluids.

Another characteristic of the sealant is the degree of swelling itundergoes in the tissue environment. Excessive swelling is undesirable,both because it creates pressure and stress locally, and because aswollen sealant gel loses tensile strength, due to the plasticizingeffect of the imbibed solvent (in this case, the solvent isphysiological fluid). Gel swelling is modulated by the hydrophobicity ofthe polymer chains. In some cases it may be desirable to derivatize thebase polymer of the sealant so that it is less hydrophilic. For example,one function of methylated collagen containing sealant is presumably tocontrol swelling of the gel. In another example, the sealant made frompenta-erythritol tetra-thiol and polyethylene glycol diacrylate can bemodified to include polypropylene glycol diacrylate, which is lesshydrophilic than polyethylene glycol. In a third example, sealantscontaining gelatin and starch can also be methylated both on the gelatinand on the starch, again to decrease hydrophilicity.

4. Suitable and Non-Suitable Sealants

Sealants suitable for purposes of this invention include the sealantsprepared from gelatin and di-aldehyde starch triggered by mixing aqueoussolutions of gelatin and dialdehyde starch which spontaneously react andgel. The gel bonds to tissue through a reaction of aldehyde groups onstarch molecules and amino groups on proteins of tissue, with anadhesive bond strength to up to 100 N/m and an elastic modulus of 8×106Pa, which is a characteristic of a relatively tough, strong material.After swelling in physiological fluids this cohesive strength declines.The gelled sealant is degraded by enzymes that cleave the peptide bondsof gelatin and the glycosidic bonds of starch.

Another acceptable sealant is made from a copolymer of polyethyleneglycol and poly-lactide or -glycolide, further containing acrylate sidechains and gelled by light, in the presence of some activatingmolecules. The linkage is formed by free-radical chemistry. The gelbonds to tissue by mechanical interlock, having flowed into tissuesurface irregularities prior to curing. The sealant degrades from thetissue by hydrolytic cleavage of the linkage between polyethylene glycolchains, which then dissolve in physiological fluids and are excreted.

The acceptable sealant made from periodate-oxidized gelatin remainsliquid at acid pH, because free aldehyde and amino groups on the gelatincannot react. To trigger gelation, the oxidized gelatin is mixed with abuffer that raises the pH, and the solution gels. Bonding to tissue isthrough aldehyde groups on the gelatin reacting with amino groups ontissue. After gelation, the sealant can be degraded enzymatically, dueto cleavage of peptide bonds in gelatin.

Still another sealant made from a 4-armed pentaerythritol thiol and apolyethylene glycol diacrylate is formed when these two neat liquids(not dissolved in aqueous buffers) are mixed. The rate of gelation iscontrolled by the amount of a catalyst, which can be a quaternary aminocompound, such as tri-ethanolamine. A covalent linkage is formed betweenthe thiol and acrylate, to form a thio-ether bond. The final gel is firmand swells very little. The tensile strength of this gel is high, about2 MPa, which is comparable to that of cyanoacrylate acceptableSuperglue. Degradation of such gels in vivo is slow. Therefore, the gelmay be encapsulated or incorporated into tissue.

Another example is the composition, preferred for use in this invention,that contains 4-armed tetra-succinimidyl ester or tetra-thiolderivatized PEG, plus methylated collagen. The reactive PEG reagents inpowder form are mixed with the viscous, fluid methylated collagen(previously dissolved in water); this viscous solution is then mixedwith a high pH buffer to trigger gelation. The tensile strength of thiscured gel is about 0.3 MPa. Degradation presumably occurs throughhydrolytic cleavage of ester bonds present in the succinimidyl esterPEG, releasing the soluble PEG chains which are excreted.

In general, a sealant useful for the purposes of this application hasadhesive, or peel strengths at least 10 N/m and preferably 100 N/cm; itneeds to have tensile strength in the range of 0.2 MPa to 3 MPa, butpreferably 0.8 to 1.0 MPa. In so-called “lap shear” bonding tests,values of 0.5 up to 4-6 N/cm² are characteristic of strong biologicaladhesives.

Such properties can be achieved by a variety of materials, both naturaland synthetic. Examples include: 1) gelatin and di-aldehyde starch(International Patent Publication Number WO 97/29715; 21 Aug. 1997); 2)4-armed penta-erythritol tetra-thiol and polyethylene glycol diacrylate(International Patent Application Number WO 00/44808; 3 Aug. 2000;example 14); 3) photo-polymerizable polyethyleneglycol-co-poly(a-hydroxy acid) diacrylate macromers (U.S. Pat. No.5,410,016; Apr. 25, 1995); 4) periodate-oxidized gelatin (U.S. Pat. No.5,618,551, Apr. 8, 1997); 5) serum albumin and di-functionalpolyethylene glycol derivatized with maleimidyl, succinimidyl,phthalimidyl and related active groups (International Patent PublicationNumber WO 96/03159, Feb. 8, 1996) and 6) 4-armed polyethylene glycolsderivatized with succinimidyl ester and thiol, plus methylated collagen,referred to as “CT3” (U.S. Pat. No. 6,312,725 B1, Nov. 6, 2001).

Various other sealant formulations are available commercially or aredescribed in the literature. However, the majority of these are notsuitable for practicing this invention for a variety of reasons.

For example, fibrin sealant is unsuitable because it interferes with theformation of cartilage.

Cyanoacrylate, or Superglue, is extremely strong but it might exhibittoxic reactions in tissue.

Un-reinforced hydrogels of various types typically exhibit tensilestrengths of lower than 0.02 MPa, which is too weak to support theadhesion required for the purpose of this application because such gelswill swell too much, tear too easily, and break down too rapidly.

It is worth noting that it is not the presence or absence of particularprotein or polymer chains, such as gelatin or polyethylene glycol, whichnecessarily govern the mechanical strength and degradation pattern ofthe sealant. The mechanical strength and degradation pattern arecontrolled by the cross-link density of the final cured gel, by thetypes of degradable linkages which are present, and by the types ofmodifications and the presence of reinforcing molecules, which mayaffect swelling or internal gel bonding.

5. Preferred Sealants

The first or second sealant of the invention must be a biologicallyacceptable, typically rapidly gelling synthetic compound havingadhesive, bonding and/or gluing properties, and is typically a hydrogel,such as derivatized polyethylene glycol (PEG) which is preferablycross-linked with a collagen compound, typically alkylated collagen.Sealant should have a tensile strength of at least 0.3 MPa. Examples ofsuitable sealants are tetra-hydrosuccinimidyl or tetra-thiol derivatizedPEG, or a combination thereof, commercially available from CohesionTechnologies, Palo Alto, Calif. under the trade name CoSeal™, describedin J. Biomed. Mater. Res (Appl. Biomater.), 58:545-555 (2001). Othercompounds suitable to be used are the rapid gelling biocompatiblepolymer compositions described in the U.S. Pat. No. 6,312,725 B1, hereinincorporated by reference. Additionally, the sealant may be two ormore-part polymers compositions that rapidly form a matrix where atleast one of the compounds is polymer, such as, polyamino acid,polysaccharide, polyalkylene oxide or polyethylene glycol and two partsare linked through a covalent bond and cross-linked PEG with methylcollagen, commercially available.

The sealant of the invention typically gels rapidly upon contact withtissue, particularly with tissue containing collagen. The second sealantmay or may not be the same as the first sealant. Both the first and thesecond is preferably a cross-linked polyethylene glycol hydrogel withmethyl-collagen, which has adhesive properties.

c) Implanting the Neo-Cartilage Construct

Next step in the method of the invention comprises implanting saidneo-cartilage into a lesion cavity formed under the second sealant orbetween two layers of sealants, said cavity either filled withneo-cartilage construct deposited therein or, optionally, with a spaceholding thermo-reversible gel (SHTG) deposited into said cavity as a solat temperatures between about 5 to about 30° C. wherein, within saidcavity and at the body temperature, said SHTG converts the sol into geland in this form the SHTG holds the space for introduction of theneo-cartilage construct and provides protection for the neo-cartilageand wherein its presence further promotes in situ formation of de novosuperficial cartilage layer covering the cartilage lesion.

The above step is versatile in that the neo-cartilage may be depositedinto said lesion cavity after the first sealant is deposited but beforethe second sealant is deposited over it or the first and second sealantsmay be deposited first and the cavity is filled with the space-holdingthermo-reversible gel for the interim period when the neo-cartilage iscultured and processed or it may be deposited into the lesion cavitywithout the first sealant and covered with the second sealant.

The neo-cartilage is either autologous or heterologous and is preparedas described above in sections I.B. a-c.

d) Removing the Space-Holding or Thermo-Reversible

from the Lesion Cavity

The neo-cartilage is deposited into the cavity either before or afterthe formation of the superficial cartilage layer. In all cases when thefirst sealant is used, the first sealant is deposited first. In oneembodiment, the neo-cartilage construct containing, typically, theheterologous neo-cartilage might be deposited on the top of the firstsealant layer and immediately covered by the second sealant layer. Insuch an instance, the neo-cartilage is left in the cavity until thesuperficial cartilage layer is formed and the neo-cartilage isintegrated into the surrounding cartilage. Then, depending on thematerial used for neo-cartilage construct, the sponge gel orthermo-reversible gelling hydrogel are left in the cavity todisintegrate.

In the instance when the two sealants are deposited first, the spacewithin the lesion cavity is optionally filled with a polymer gel, suchas the space-holding thermo-reversible gel. Such gel is left in thecavity until the neo-cartilage construct is cultured, processed andready to be implanted. Since such thermo-reversible gel might or mightnot be completely or partially degraded during this time, it may beremoved from the cavity by cooling the lesion to about 5° C. at whichtemperature the gel becomes a sol, and by removing said sol from thecavity, for example, by injection. Using the same process, that is bycooling the solid gel of the neo-cartilage, the process may be reversedfor introduction of the neo-cartilage construct into said lesion cavitywherein, after the sol is warmed into the body temperature, the sol isconverted into a solid gel.

Thus, the primary premise of this process is that the removal and/orintroduction of the space holding gel or introduction of neo-cartilageconstruct proceeds at the cold temperature where the composition is inthe sol state and converts into solid gel at warmer temperatures. Inthis way the gel may be removed from the cavity as the sol after theneo-cartilage integration and formation of superficial cartilage layer.

e) Generation of the Superficial Cartilage Layer

A combination of the neo-cartilage construct comprising theneo-cartilage suspended in the thermo-reversible gel or support matrixembedded with chondrocytes with the adhesive polymeric second sealantleads to overgrowth and complete or almost complete sealing of thelesion cavity.

In alternative, depending on the surface chemistry of thethermo-reversible gel, the superficial layer could grow directly overthe neo-cartilage construct if such surface chemistry is propitious tosuch growth.

Typically, a biologically acceptable second sealant, preferably across-linked PEG hydrogel with methyl collagen sealant, is depositedeither over the neo-cartilage construct implanted into the lesion cavityor is deposited over the lesion before the neo-cartilage construct isdeposited therein. The second sealant acts as an initiator for formationof the superficial cartilage layer which in time completely overgrowsthe lesion. The superficial cartilage layer in several weeks or monthscompletely covers the lesion and permits integration of theneo-cartilage of the neo-cartilage construct or chondrocytes embeddedwithin the support matrix into the native surrounding cartilagesubstantially without formation of fibrocartilage.

Formation of the superficial cartilage layer is a very important aspectof the healing of the cartilage and its repair and regeneration.

IV. In Vivo Studies in Swine of Weight-Bearing Region of the Knee

The method according to the invention was tested and confirmed in invivo studies wherein the generation of the superficial cartilage layerhas been confirmed in a three month study performed in a swine model inorder to evaluate porcine neo-cartilage construct integration into thesurrounding cartilage.

The neo-cartilage construct prepared according to the method of theinvention was implanted into an artificially generated lesion in a pig'sknee. Detailed conditions of the study are described in Example 8.Results of this study are illustrated in FIGS. 10, 11 and 12 depictinghistological evaluation using Safranin-O staining method of artificiallycreated cartilage lesions.

Briefly, the study comprised of an open arthrotomy of the right kneejoint performed on all animals. A biopsy of the cartilage was obtained.Chondrocytes were isolated from the cartilage biopsy and cultured withina collagen matrix in a Tissue Engineering Support System (TESS™) asdescribed in detail above to produce porcine neo-cartilage construct forsubsequent implantation.

A defect was created in the medial femoral condyle of the right knee.This defect, which served as a control, was not implanted with theneo-cartilage construct. The empty defect is seen in FIG. 10A. Followingsurgery, the joint was immobilized with an external fixation device fora period of about two weeks. Three weeks after the arthrotomy on theright knee was performed, an open arthrotomy was performed on the leftknee and the same defects were created in this medial femoral condyle.The porcine neo-cartilage was implanted within defects in this kneewhich was similarly immobilized. The porcine implant site is seen inFIG. 10B which also show initiation of formation of a superficialcartilage layer two weeks after implantation.

The operated sites were periodically viewed via arthroscopy at monthlyintervals. Subsequently, approximately 3 months after porcineneo-cartilage implantation, animals were euthanized and joints harvestedand prepared for histological examination. The implanted sites wereprepared and examined histologically. Comparison of FIG. 11 (control atfour months after arthrotomy) and FIG. 12 shows test knee three monthfollowing arthrotomy and neo-cartilage implantation according to theinvention. This figure shows that in the control knee there is a visibleformation of fibrocartilage. In the test group (FIGS. 12A-12D), theimplanted porcine neo-cartilage construct resulted in production ofdense regenerating hyaline cartilage and in the same test group, therewas clearly visible cell integration (FIGS. 12C and 12D) and formationof the superficial cartilage layer (FIGS. 12A and 12B).

FIGS. 11A-11C thus shows the control lesion at 4 months following thesurgery without a treatment with the neo-cartilage construct. Noticeablein FIG. 11A is the proliferation of undesirable fibrocartilage withinthe defect site. Also seen is synovial tissue that has infiltrated intothe subchondral space.

FIGS. 12A-12D, on the other hand, show that after 3 months postimplantation in a weight bearing region of the knee, the porcineneo-cartilage has produced dense hyaline-like cartilage and hasintegrated with the host cartilage laterally and at the interface of thesubchondral bone.

Additionally, FIG. 12A shows a formation of regenerated hyaline-likecartilage in the implant site; FIG. 12B shows the beginning ofintegration between the porcine neo-cartilage and the native cartilagelaterally and at the subchondral bone. FIG. 12C shows alreadyregenerated hyaline-like cartilage and FIG. 12D shows chondrocytesintegration into the surrounding native cartilage.

The porcine neo-cartilage was delivered to the defect by implantation ofneo-cartilage construct between two layers of sealant. The newly formedsuperficial cartilage layer formed over the defect at three monthsfollowing the implantation is clearly visible.

FIG. 12 thus shows and confirms that 3 months after implantation in aweight-bearing region of the knee, the porcine-NeoCart™ has produceddense hyaline-like cartilage and has integrated with the host cartilagelaterally and at the interface of the subchondral bone.

These results confirm that the damaged, injured, diseased or agedcartilage may be repaired by using neo-cartilage implants preparedaccording to the algorithm of the invention.

V. Human Osteoarthritic Cartilage

Articular cartilage is a unique tissue with no vascular, nerve, orlymphatic supply. The lack of vascular and lymphatic circulation may beone of the reasons why articular cartilage has such a poor intrinsiccapacity to heal, except for formation of fibrous or fibrocartilaginoustissue. Unique mechanical functions of articular cartilage are neverreestablished spontaneously after a significant injury or disease, suchas osteoarthritis (OA).

In osteoarthritis, disruption of the structural integrity of the matrixby the degeneration of individual matrix proteins leads to reducedmechanical properties and impaired function.

Currently, the only available treatment of severe osteoarthritis of theknee is a total knee replacement in elderly patients. In young andmiddle aged patients, however, there is no optimal treatment.

In order to evaluate suitability of the current invention for treatmentof osteoarthritis, studies using algorithm of the invention including aTESS culture system using neo-cartilage scaffold construct and algorithmof the invention (hydrostatic pressure and medium perfusion) on human OAchondrocytes, cell proliferation and extracellular matrix accumulationin OA chondrocytes was investigated.

Results are seen in Table 5 and in FIGS. 13-15. TABLE 5 PressureConditions Total DNA content In TESS days S-GAG Production (μg/cellGroup Type of In (μg/cell construct) construct) (n = 7) Pressure Time InIncubator culture (Mean ± SD) (Mean ± SD) Initial — — 0 day 0 day 19.23± 0.87 1.88 ± 0.40 Control — — 21 days 21 days 23.81 ± 2.61 2.34 ± 0.32Cy-HP#1 0.5 MPa 7 days 14 days 21 days *29.53 ± 1/60  2.33 ± 0.12 CyclicCy-HP#2 0.5 MPa 14 days  7 days 21 days *34.39 ± 0.99  2.35 ± 0.09Cyclic Const-HP 0.5 MPa 7 days 14 days 21 days 26.94 ± 5.14 **2.65 ±0.28  Constant(*p < 0.05, compared to Control in S-GAG production)(**p < 0.05, compared to Initial in DNA content)

In the TESS processor, the medium flow rate was 5 μl/min and thehydrostatic pressure was applied as indicated. Two cell matrices fromeach group were harvested for histological analysis.

As seen in Table 5 and FIG. 13A, S-GAG production in cell constructssubjected to cyclic hydrostatic pressure with medium perfusion wassignificantly greater than those subjected to atmospheric pressure(control). Especially, S-GAG production (μg/cell construct) wassignificantly increased (144%) for Cy-HP#2 where the cyclic hydrostaticpressure was used for 14 days followed by 7 days of static atmosphericpressure compared to control.

FIG. 13B shows DNA content index with corresponding results presented inTable 5 for DNA, likewise showing increased production of DNA. Increasein DNA content index in cell constructs using the neo-cartilageconstruct subjected to constant hydrostatic pressure was clearly shownin a comparison to initial level. DNA level in cell constructs subjectedto constant hydrostatic pressure with medium perfusion was significantlyincreased to 142% compared to initial DNA level index.

FIGS. 14A-14E show histological evaluation of cell constructs bySafranin-O. FIG. 14A shows S-GAG accumulation at day 0 (initial). FIG.14B shows accumulation of S-GAG on day 21 in cell constructs subjectedto atmospheric pressure (control). FIG. 14C shows accumulation of S-GAGon day 21 in cell constructs subjected to 7 days of cyclic hydrostaticpressure (Cy-HP#1) followed by 14 days of the atmospheric pressure. FIG.14D shows accumulation of S-GAG on day 21 in cell constructs subjectedto 14 days of cyclic hydrostatic pressure (Cy-HP#2) followed by 7 daysof to atmospheric pressure. FIG. 14E shows accumulation of S-GAG on day21 in cell constructs subjected to 7 days of constant hydrostaticpressure (Constant-HP) followed by 14 days of atmospheric pressure. Thegreater S-GAG accumulation in both cell constructs subjected to cyclichydrostatic pressure (7 and 14 days) is evident from the increaseddensity of the photomicrograph clearly visible in the FIGS. 14C and 14D.

These results demonstrate that hydrostatic pressure combined with amedium perfusion promotes both cell proliferation and neo-cartilagephenotypic activity, that is, cartilage extracellular matrix production,in the scaffold neo-cartilage constructs seeded with human OAchondrocytes. This evidence confirms that the algorithm of inventionusing TESS culture system and hydrostatic pressure combined with mediumperfusion regenerates human OA chondrocytes and transforms the OAcartilage into the healthy hyaline cartilage.

VI. Method for Treatment of Cartilage Lesions

The method for treatment of damaged, injured, diseased or aged cartilageaccording to the invention is suitable for healing of small lesion dueto acute injury as well as healing of the large lesions caused byosteoarthritis or other joint degenerative diseases and/or transformingthe diseased OA cartilage into the healthy hyaline cartilage.

The method generally encompasses five novel features, namely, employinga biologically acceptable thermo-reversible polymer gel as a carriersupport matrix for neo-cartilage generated from autologous chondrocytes,producing the autologous neo-cartilage by a process of the invention,employing a biologically acceptable thermo-reversible gel as aspace-holding means for the interim period when the autologousneo-cartilage is produced, depositing one or two adhesive sealants tothe lesion and, following depositing the sealants and implantation ofthe neo-cartilage within a cavity generated thereby, a formation of thesuperficial cartilage layer covering the lesion and protecting theintegrity of the neo-cartilage deposited therein.

The method generally comprises steps:

a) debriding an articular cartilage lesion and during the debridingharvesting a small quantity (50-4000 mg) of non-osteoarthritic hyalinecartilage;

b) fabrication and processing of the neo-cartilage construct accordingto the above described procedures;

c) preparing the lesion for implantation of the neo-cartilage constructby depositing the one or two sealant layers, the first (optional) at thebottom of the lesion and the second one over and on the top of thelesion, and, using all variation already described above, depositingeither the neo-cartilage construct within the cavity formed below thetop sealant and/or between the two sealant layers or depositing thespace holding thermo-reversible polymer gel into the cavity between thetwo layers to uphold the integrity of the cavity in the interim when theneo-cartilage construct is being prepared;

d) implanting the neo-cartilage construct into said cavity formedbetween the two sealant layers to allow for integration of theneo-cartilage into the surrounding native intact cartilage and formationof the superficial cartilage layer; and

e) optionally removing the space holding polymer gel from the cavitybefore the neo-cartilage implantation.

In the alternative method for treatment, expanded and differentiatedchondrocytes may be deposited directly into a joint lesion in a suitabletypically thermo-reversible gelation hydrogel solution.

There are several advantages of the current method. First, the method isvery versatile and any of the variations may be advantageously utilizedfor treatment of a specific injury, damage, aging or disease.

The method permits generation of autologous neo-cartilage by providingalternative means for maintaining a space between two sealant layersuntil the autologous neo-cartilage is prepared. The method permitsgeneration of more dense neo-cartilage and three-dimensional expansionof chondrocytes and extracellular matrix.

The deposition of the second top sealant layer resulting in formation ofsuperficial cartilage layer constitutes a substitute for synovialmembrane and provides the outer surface of healthy articular cartilageovergrowing, protecting, containing and providing critical metabolicfactors aiding in growth and incorporation of autologous neo-cartilagein the lesion.

Deposition of the first bottom sealant layer protects the integrity ofthe lesion after cleaning during surgery and prevents migration ofsubchondral and synovial cells and cell products thereby creating milieufor formation of healthy hyaline cartilage from the neo-cartilage andalso preventing formation of the fibrocartilage.

The method further permits deposition of the space-holding gel orthermo-reversible polymer gel to be deposited whether alone or withsuspended processed neo-cartilage into the lesion at temperature between5 and 30° C. as a sol. Selection of thermo-reversible gel may be crucialas certain TRGH may function as a promoter for growth of the superficialcartilage layer without a need to apply the second sealant.

The method further permits said thermo-reversible hydrogel be enhancedwith hyaluronic acid, typically added in about 5 to about 50%,preferably about 20% (v/v), wherein such hyaluronic acid acts as anenhancer of the matrix-forming characteristics of the gel and to act asa hydration factor in the synovial space in general and within thelesion cavity in particular.

Additionally, the gel acts as a slow-release unit for hyaluronic acid,greatly increasing a period of hydration within the cavity and also as asubstrate for formation of the superficial cartilage layer and it canalso be conveniently removed, if needs be, by cooling the lesion so thatthe solid gel formed at 37° C. is converted to sol and can be removed byinjection or otherwise.

For treatment of the cartilage, a subject is treated, according to thisinvention, with a prepared autologous or heterologous neo-cartilage orneo-cartilage construct implanted into the lesion, the neo-cartilage orthe construct is left in the lesion for two-three months and typically,it does not need any further intervention as during these three months,the neo-cartilage is fully integrated into the native cartilage andbecomes a fully functional cartilage covered with a superficialcartilage layer which eventually grows into or provides the same type ofsurface as a synovial membrane of the intact joint.

Finally, the diseased, osteoarthritic cartilage may be fully replaced bythe regenerated hyaline-like cartilage when processed according to thealgorithm of this invention.

The algorithm and/or implantation protocol may assume any variationdescribed above or possible within the realm of this invention. It isthus intended that every and all variations in the treatment protocol(algorithm of the cartilage) are within the scope of the currentinvention.

EXAMPLE 1 Isolation of Chondrocytes from Source Tissue

This example describes the procedure used for isolation of chondrocytesfrom swine cartilage.

Chondrocytes were enzymatically isolated from cartilage harvested understerile conditions from the hind limbs of 6-month old swine. The femurwas detached from the tibia and the trachea head exposed. Strips ofcartilage were removed from the trachea using a surgical blade.

The cartilage was minced, digested in a 0.15% collagenase type Isolution in DMEM/Nutrient Mixture F-12 (DMEM/F-12) 1:1 mixture with 1%penicillin-streptomycin (P/S) and gently rotated for 18 hours at 37° C.Chondrocytes were collected and rinsed twice by centrifugation at 1500rpm for 5 min. Chondrocytes were re-suspended in DMEM/F-12 containing 1%penicillin-streptomysin and 10% FBS.

Chondrocytes were expanded for about 5 days at 370C.

EXAMPLE 2 The Production of Human Neo-Cartilage Construct

This example describes conditions for production of neo-cartilage forhuman use.

The patient undergoes arthroscopic biopsy of a small (200-500 mg) pieceof healthy cartilage from the ipsilateral knee. The biopsy is taken fromthe non-weight bearing portion of the femoral condyle or from thefemoral notch as deemed most appropriate for the patient. The biopsysample is placed into a sterile, non-cytotoxic, non-pyrogenic specimencontainer which is packaged and shipped to the laboratory.

At the laboratory the biopsy sample is examined against acceptancecriteria and then transferred to the chondrocyte isolation and expansionarea. Samples from the biopsy specimen transport buffer are tested forsterility and for mycoplasma. The expanded chondrocytes are suspended inVITROGEN® gellable collagen solution, commercially available fromCohesion Corp., Palo Alto, Calif. A pre-formed collagen sponge (22×22 mmsquare and 2-4 mm in thickness, wherein the thickness depends on thethickness of patient's cartilage), commercially available from KokenCo., Japan or honeycomb matrix produced according to this invention isplaced into the resulting chondrocyte suspension which absorbs thechondrocyte/collagen suspension into this matrix.

The resulting chondrocyte-loaded matrix is warmed to 37° C. to gel theVITROGEN in order to spatially secure the chondrocytes within thesupport matrix. The loaded support matrix is then placed into TissueEngineering Support System (TESS™) culture unit. Typical time for cellexpansion from removal of a biopsy sample to placement of thechondrocyte loaded culture matrix in the TESS™ culture unit is 10-40days. Within the TESS™ culture unit, cyclic or constant hydrostaticpressure is used to induce the chondrocytes to begin growing andexpressing their cartilage generating program for about 1 hour to about30 days.

The still developing new cartilage is transferred to a constant, restingculture phase. The neo-cartilage production process requires a minimumtime of 10 days in resting culture. After this minimum 10-day period theneo-cartilage, hereinafter called neo-cartilage construct, undergoesfinal inspections and is packaged for return to the clinic to beimplanted. At the time of release, tests for sterility, endotoxin, andmycoplasma contamination must be negative for microbial and mycoplasmacontamination and must show ≦0.5 EU/ml of endotoxin.

EXAMPLE 3 Preparation of Support Matrices

This example illustrates preparation of the cellular support matrix,also called the TESS matrix.

300 grams of a 1% aqueous atelocollagen solution (VITROGEN®), maintainedat pH 3.0, is poured into a 10×20 cm tray. This tray is then placed in a5 liter container. A 50 ml open container containing 30 ml of a 3%aqueous ammonia solution is then placed next to the tray, in the 5 literchamber, containing 300 grams of said 1% aqueous solution ofatelocollagen. The 5 liter container containing the open trays ofatelocollagen and ammonia is then sealed and left to stand at roomtemperature for 12 hours. During this period the ammonia gas, releasedfrom the open container of aqueous ammonia and confined within thesealed 5 liter container, is reacted with the aqueous atelocollagenresulting in gelling said aqueous solution of atelocollagen.

The collagenous gel is then washed with water overnight and,subsequently, freeze-dried to yield a sponge like matrix. This freezedried matrix is then cut into squares, sterilized, and stored under asterile wrap.

Alternatively, the support matrix may be prepared as follows.

A porous collagen matrix, having a thickness of about 4 mm to 10 mm, ishydrated using a humidity-controlled chamber, with a relative humidityof 80% at 25° C., for 60 minutes. The collagen material is compressedbetween two Teflon sheets to a thickness of less than 0.2 mm. Thecompressed material is then cross-linked in a solution of 0.5%formaldehyde, 1% sodium bicarbonate at pH 8 for 60 minutes. Thecross-linked membrane is then rinsed thoroughly with water, andfreeze-dried for about 48 hours. The dense collagen barrier has an innerconstruction of densely packed fibers that are intertwined into amulti-layer structure.

In alternative, the integration layer is prepared from collagen-baseddispersions or solutions that are air dried into sheet form. Drying isperformed at temperatures ranging from approximately 4 to 40° C. for aperiod of time of about 7 to 48 hours.

EXAMPLE 4 Seeding Cells in the TESS Matrix

This example describes procedures used for seeding cells in the TESSmatrix.

Isolated chondrocytes were incubated for a period of five days at 37° C.in a standard incubator. Cells were then collected by trypsinization.

A cell suspension of 150,000 cells in 18 μl of VITROGEN solution wasseeded per matrix having an approximate volume of 19 μl, with ninematrices per group. The seeded matrix (collagen sponge 4 mm in diameterand 1.5 mm in thickness) may be scaled-up to an increased volume, whereapproximately 1 μl of the above described cell suspension is seeded in 1μl of matrix. The control group matrices were incubated in a 37° C.incubator and the test group was incubated in the TESS.

In alternative set-up, isolated chondrocytes were incubated for a periodof five days at 37° C. in a standard incubator. Cells were thencollected by trypsinization. A cell suspension of 300,000 cells in 18 μlof VITROGEN solution was seeded per matrix having an approximate volumeof 19 μl with eight matrices per group.

EXAMPLE 5 Effect of Cyclic Hydrostatic Pressure

This example describes procedures used for determination of effect ofcyclic hydrostatic pressure in vitro formation of chondrocyte-seededsupport matrices.

Swine articular chondrocytes (sACs) were enzymatically isolated fromcartilage with type I collagenase. The cells were suspended in collagen(VITROGEN) as described above and wicked into the honeycombed spongeelement of the cellular support matrix. The cells seeded in the supportmatrix were incubated at 37° C., 5% CO₂ and 20% O₂. After 24 hours, someof these cells matrices were transferred to the TESS™ processor andincubated at 0.5 or 3.0 MPa cyclic or constant hydrostatic pressure withmedium perfusion (0.05 ml/min) as described above for 6 or 7 daysfollowed by a 12 or 14 day resting phase. The control group comprised ofchondrocytes seeded in matrices incubated for 18 or 21 days atatmospheric pressure, at 37° C., 5% CO₂ and 20% or 2% O₂.

At the end of the culture period (18 or 21 days), the matrices wereharvested for biochemical and histological analysis. For biochemicalanalysis, sulfated glycosaminoglycan (S-GAG) production was measuredusing a modified dimethylmethylene blue (DMB) microassay.

Two matrices from each group were harvested for histological analysis.

EXAMPLE 6 Effect of Medium Flow Rate on Extracellular Matrix

Accumulation of Chondrocytes in Collagen Sponges This example describedconditions used to determine effect of medium flow on production andaccumulation of extracellular matrix by chondrocytes seeded intocollagen sponges.

Chondrocyte Isolation

Swine legs were obtained from a local abattoir. Within 4-6 hours afterslaughter, cartilage was harvested under sterile conditions from thetrochlea of the hind limbs. The cartilage was minced and digested in0.15% collagenase type I in DMEM/F-12 containing 1%penicillin-streptomycin (P/S) for 18 hours at 37° C. Isolated swinearticulate chondrocytes (sACs) were collected, rinsed, and resuspendedin DMEM/F-12 supplemented with 10% fetal bovine serum (FBS) and 1% P/S.sACs then were expanded for 5 days at 37° C.

Cell Seeding in Collagen Sponges

sACs were harvested with Trypsin EDTA and cell viability was measured bytrypan-blue exclusion. Three hundred thousand sACs were suspended in 30μl of a neutralized 0.25% collagen solution (VITROGEN®, Cohesion Corp.,Palo Alto, Calif.), and the suspension was absorbed into a collagensponge, 4 mm in diameter and 2 mm in thickness, commercially availablefrom Koken Co., Japan. Seeded sponges were pre-incubated for 1 hour at37° C. to gel the collagen, followed by incubation in culture medium at37° C. in 5% CO₂.

Tissue Engineering Support System (TESS™) Culture

Following the incubation, the seeded sponges were transferred to andcultured in the Tissue Engineering Support System (TESS™) processor. Toevaluate the effect of medium perfusion rate, sponges were subjected tomedium perfusion at 5 μl/min or 50 μl/min. Cyclic hydrostatic pressure(Cy-HP) 0-0.5 MPA pressure at 0.5 Hz applied was for 6 days. Somesponges were incubated under constant conditions at atmospheric pressureand no perfusion at 37° C. for a total of 18 days in culture. Spongesharvested 24 hours after seeding with cells (day 0) served as an initialcontrol.

Histological and Biochemical Analysis

Cell constructs were harvested after 6 and 18 days of culture.

For histological evaluation, 4% paraformaldehyde-fixed, paraffinsections were stained with Safranin-O (Saf-O) and Type II collagenantibody.

For biochemical analysis, seeded sponges were digested in papain at 60°C. for 18 hours and DNA content was measured using the Hoechst 33258 dyemethod. Sulfated glycosaminoglycan (S-GAG) accumulation was measuredusing a modified dimethylmethylene blue (DMB) microassay.

EXAMPLE 7 Biochemical and Histological Assays

This example describes assays used for biochemical and histologicalstudies (DMB assay).

Biochemical (DMB) Assay

At the end of the culture six matrices from each group were used in thebiochemistry assay.

The matrices were transferred to microcentrifuge tubes and digested in300 μl of papain (125 μg/ml in 0.1 M sodium phosphate, 5 mM disodiumEDTA, and 5 mM L-cysteine-HCl) for 18 hours at 60° C. GAG production inthe matrices was measured using a modified dimethylene blue (DMB)microassay with shark chondroitin sulfate as a control Connective TissueResearch, 9: 247-248 (1982).

DNA content was determined by Hoechst 33258 dye method according toAnal. Biochem., 174:168-176 (1988).

Histological Assay

The remaining matrices from each group were fixed in 4%paraformaldehyde. The matrices were processed and embedded in paraffin.10 μm sections were cut on a microtome and stained with Safranin-O (SafO).

EXAMPLE 8 Evaluation of Porcine Neo-Cartilage Integration in a SwineModel

This example describe the procedure and results of study performed forevaluation of integration of porcine neo-cartilage in a swine model.

An open arthrotomy of the right knee joint was performed on all animals,and a biopsy of the cartilage was obtained.

Chondrocytes were isolated from the cartilage biopsy and cultured withina collagen matrix in a Tissue Engineering Support System (TESS™) toproduce porcine-Neocart for subsequent implantation.

A defect was created in the medial femoral condyle of the pig's rightknee. This defect (control) was not implanted with porcine-NeoCart™.Following surgery, the joint was immobilized with an external fixationconstruct for a period of about two weeks. Three weeks after thearthrotomy on the right knee was performed, an open arthrotomy wasperformed on the left knee and defects were created in this medialfemoral condyle. The porcine-NeoCart™ was implanted within the defect(s) in this knee which was similarly immobilized. The operated siteswere subsequently viewed via arthroscopy two weeks after implantation ordefect creation and thereafter at monthly intervals. Animals wereeuthanized and the joints harvested and prepared for histologicalexamination approximately 3 months after porcine-NeoCart™ implantation.The implanted sites were prepared and examined histologically.

Results are seen in FIGS. 10-12. FIG. 10 shows results of thearthroscopic examination. The empty defect is seen in FIG. 10A. Theporcine NeoCart™ implant site is seen in FIG. 10B which also showsstill-evident absorbable sutures and the superficial cartilage layergrowing over the porcine NeoCart™.

EXAMPLE 9 Protocol for In Vivo, Ex Vivo or In Vitro Growth of PorcineNeo-Cartilage

Autologous porcine chondrocytes are seeded into the cellular supportmatrix and incubated under cyclic hydrostatic pressure at 37° C. and 5%CO₂. Cyclic hydrostatic pressure is either 0.5 or 3.0 MPa at 0.5 Hz. Theduration of said cyclic pressure is approximately 6 days followed by aresting phase of 12 days in an incubator maintained at 37° C. atatmospheric pressure. At the end of this resting phase, the matriceswere harvested for biochemical and histological analysis.

In the alternative protocol, the algorithm for the growth cells of invivo and in vitro, the application of hydrostatic pressure is used onisolated in situ cartilage, or application of hydrostatic pressure forabout 1-8 hours followed by about 16-23 hours of recovery period.

EXAMPLE 10 Regeneration of Human Chondrocytes

This example describes the procedure used for regeneration of humanchondrocytes.

Chondrocytes from osteoarthritic (OA) patients (40 years old) wereexpanded for 18 days in monolayer culture at 37° C. and suspended inVITROGEN® (300,000 cells/30 fÊl). The cell suspension was absorbed intoa support matrix, usually a collagen honeycomb sponge (4 mm in diameterand 2 mm in thickness, Koken Co., Japan). The cell constructs wereincubated in culture medium supplemented with 10% FBS and 1% ITS(insulin-transferrin-sodium selenite, Sigma) at 37° C., 5% CO₂ and 20%O₂, at 0.5 MPa cyclic hydrostatic pressure (Cy-HP) or 0.5 MPa constanthydrostatic pressure (Constant-HP) for 7 or 14 days in the TESS™processor followed by incubation for 7 or 14 days at atmosphericpressure for 7 or 14 days in an CO₂ incubator at 37° C. The remainingcell constructs compromising the control group were incubatedatmospheric pressure for 21 days at 37° C., 5% CO₂ and 20% O₂.

Before starting the culture, some cell constructs were harvested forbiochemical and histological analysis as an initial condition. At theend of the culture period, the cell constructs were harvested forbiochemical and histological analysis. Sulfated glycosaminoglycanproduction was measured using a modified dimethylmethylene blue (DMB)micro assay. Cell proliferation was measured using a modified HoechstDye DNA assay. Formation of neo-tissue was analyzed by Safranin-O

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 10. A method forfabrication of a three-dimensional neo-cartilage construct for in situimplantation into a cartilage lesion, said method comprising steps: a)preparing a support matrix structure; b) harvesting a piece of cartilageand isolating chondrocytes, or obtaining chondrocytes or cells capableof differentiation into chondrocytes, from a donor; c) culturing andexpanding said chondrocytes or said cells; d) suspending the expandedchondrocytes or cells in a suspension fluid; e) incorporating saidsuspended chondrocytes or cells into said support matrix structure; andf) propagating said chondrocytes into a three-dimensional neo-cartilageconstruct using an algorithm of the invention, wherein said algorithmcomprises subjecting said chondrocytes or cells to a cyclic or constanthydrostatic pressure from about zero to about 10 MPa at from about 0.01to about 1 Hz, to atmospheric pressure or to non-pressure conditions,for from about 1 to about 24 hours a day, for about 1 to about 90 daysat a rate of a medium perfusion from about zero to about 500 μL/min, atan oxygen concentration from about zero to about 20% and a carbondioxide concentration of about zero to about 5%.
 11. The method of claim10 wherein the support matrix structure is prepared from a materialselected from the group consisting of collagen, a Type I collagen, aType II collagen, a Type IV collagen, a cell-contracted collagencontaining a proteoglycan, a cell-contracted collagen containing aglycosaminoglycan, a cell-contracted collagen containing a glycoprotein,gelatin, agarose, hyaluronin, fibronectin, laminin, a bioactive peptidegrowth factor, a cytokine, elastin, fibrin, a synthetic polymeric fibermade of a polylactic acid, a synthetic polymeric fiber made of apolyglycolic acid, a synthetic polymeric fiber made of a polyamino acid,polycaprolactone, a polyamino acid, a polypeptide gel, a hydrogel, asol-gel, a polymeric sol-gel, a thermo-reversible gelation hydrogel(TRGH), a polymeric TRGH, a copolymer thereof and a combination thereof.12. The method of claim 11 wherein said support matrix structure isprepared from the Type I collagen, Type II collagen or Type IV collagen,or from a sol-gel, a polymeric sol-gel, a TRGH or a polymeric TRGH. 13.The method of claim 12 wherein said support matrix structure is preparedfrom Type I collagen, Type II collagen or Type IV collagen andfreeze-dried or lyophilized into a collagen sponge, porous scaffold or ahoneycomb.
 14. The method of claim 11 wherein said support matrixstructure is prepared from the polymeric thermo-reversible gellinghydrogel (TRGH) or the polymeric sol-gel hydrogel.
 15. The method ofclaim 12 wherein the suspension fluid for said cultured and expandedchondrocytes or said cells is the gel, polymeric sol-gel or TRGH andwherein said chondrocytes are incorporated into said support matrixsuspended in said suspension fluid.
 16. The method of claim 15 whereinthe support matrix structure is the collagen sponge, porous scaffold orhoneycomb and wherein said chondrocytes or said cells are incorporatedinto said sponge, porous scaffold or honeycomb, in the TRGH suspensionfluid.
 17. The method of claim 16 wherein said chondrocytes or saidcells are suspended and incorporated into said sponge at a density offrom lower than 3 to up about 15 millions cells/ml of the suspensionfluid.
 18. The method of claim 17 wherein the cyclic hydrostaticpressure is from about 0.05 MPa to about 3 MPa above atmosphericpressure at from about 0.1 to about 0.5 Hz, wherein the constanthydrostatic pressure is from about zero to about 3 MPa, wherein the timefor applying the hydrostatic pressure is from about 1 to about 24 hoursper day for from about one day to about 14 days, wherein saidhydrostatic pressure is preceded or followed by a period of zero toabout 24 hours per day of a static atmospheric pressure for from aboutone day to about ninety days, wherein the flow rate is from about 1μL/min to about 500 μL/min, wherein the cell density is lower or about 3millions per 1 ml of the suspending fluid, wherein the oxygenconcentration is about 20% and wherein the carbon dioxide concentrationis zero.
 19. The method of claim 18 wherein the support matrix constructhas pores from about 50 μm to about 500 μm.
 20. The method of claim 19wherein said hydrostatic pressure is preceded or followed by a period ofabout zero to about 28 days of atmospheric pressure.
 21. The method ofclaim 19 wherein the support matrix construct has pores from about 100μm to about 300 μm.
 22. The method of claim 19 wherein the supportmatrix construct has pores about 100 μm.
 23. A method for preparation ofa neo-cartilage construct suitable for in situ implantation into acartilage lesion, said construct comprising a cultured differentiatedautologous or heterologous chondrocytes or cells which could bedifferentiated into chondrocytes, incorporated into a support matrix andsubjected to an algorithm of the invention, said construct prepared by amethod comprising steps a) preparing a support matrix structure; b)harvesting a piece of cartilage and isolating chondrocytes, or obtainingchondrocytes or cells capable of differentiation into chondrocytes, froma donor; c) culturing and expanding said chondrocytes or said cells; d)suspending the expanded chondrocytes or cells in a suspension fluid; e)incorporating said suspended chondrocytes or cells into said supportmatrix structure; and f) propagating said chondrocytes intothree-dimensional neo-cartilage construct using an algorithm comprisingsubjecting said propagated chondrocytes or cells within theneo-cartilage construct to a cyclic or constant hydrostatic pressurefrom about zero to about 10 MPa at from about 0.01 to about 1 Hz,preceded or followed by a period of time where said cells are subjectedto an atmospheric pressure or to non-pressure conditions, for from about1 to about 24 hours a day for about zero to about 90 days at a rate of amedium perfusion from zero to about 500 μL/min at an oxygenconcentration from about zero to about 20% and a carbon dioxideconcentration of zero to about 5% wherein a cell density of suspendedchondrocytes or cells in the suspension fluid is from below 3 millionsto about 60 millions.
 24. The method of claim 23 wherein the supportmatrix is a sponge, porous scaffold or a hydrogel prepared from amaterial selected from the group consisting of a Type I collagen, a TypeII collagen, a Type IV collagen, a cell-contracted collagen containingproteoglycan, a cell-contracted collagen containing glycosaminoglycan, acell-contracted collagen containing a glycoprotein, gelatin, agarose,hyaluronin, fibronectin, laminin, a bioactive peptide growth factor,cytokine, elastin, fibrin, a synthetic polymeric fiber made of apolylactic acid, a synthetic polymeric fiber made of a polyglycolicacid, a synthetic polymeric fiber made of a polyamino acid,polycaprolactone, a polypeptide gel, a polymeric thermo-reversiblegelation hydrogel (TRGH), a copolymer thereof and a combination thereof.25. The method of claim 24 wherein the support matrix is a sponge orporous scaffold prepared from the TRGH.
 26. The method of claim 25wherein the hydrostatic cyclic pressure is from about 0.05 MPa to about3 MPa at 0.1 to about 0.5 Hz or constant pressure is from about zero toabout 3 MPa above atmospheric pressure and wherein such pressure isapplied for about 7 to about 28 days.
 27. The method of claim 26 whereinsaid hydrostatic pressure is preceded or followed by a period of aboutone to about seven days of atmospheric pressure.
 28. The method of claim27 wherein said support matrix has pores from about 5 to about 500 μm.29. The method of claim 27 wherein said support matrix has pores fromabout 100 to about 300 μm.